Systems for Factor VIII Processing and Methods Thereof

ABSTRACT

The present invention provides methods of reducing nonprocessed Factor VIII or a chimeric polypeptide comprising Factor VIII comprising co-transfecting in a host cell a polynucleotide encoding Factor VIII with a polynucleotide encoding a protein convertase, where the endogenous processing enzymes of the host cell are insufficient to convert all of the Factor VIII to its processed isoform; expressing a proprotein convertase from a second polynucleotide in the host cell; and reducing the nonprocessed Factor VIII by processing with said proprotein convertase.

BACKGROUND OF THE INVENTION

Proprotein convertases, also known as prohormone convertases, neuroendocrine convertase, or Proprotein Convertase Subtilisin/Kexin Type (PCSK), are capable of cleaving precursor proteins. The precursor proteins that are known to be cleaved by proprotein convertases include growth factors and hormones, receptors, and bacterial endotoxins. Nakayama, K., Biochem. J. 327: 625-635 (1997). The first proprotein processing protease discovered was Kex2 protease from S. cerevisiae. Rockwell et al., Chem. Rev. 102: 4525-4548 (2002). Kex2 homologues identified in mammalian cells include PC1/3, PC2, PACE4, PC4, PC5/PC6, and PC7. See id.

Each member of the convertase family exhibits a unique tissue distribution; different cell types were found to express individual combinations of these enzymes. PC4 is limited to testicular germ cells. Seidah et al., Mol. Endocrinol. 6(10): 1559 (1992). PC1/3 and PC2 are restricted to endocrine and neuroendocrine tissues. Seidah et al., NIDA Res. Monogr. 126:132 (1992). PACE 4 and PC5 are expressed in several tissues, while PC7 exhibits an even more common tissue distribution. Furin is expressed ubiquitously.

The cellular sublocalisation of the endoproteases is an important determinant for their physiological function, PC1/3, PC2, and PC5A, an isoform of PC5/6, are involved in the processing of pro-hormones and neuropeptide precursors which are secreted in a regulated manner. PC1/3 and PC2 were shown to cleave neuroendocrine-specific proinsulin (Smeekens et al., Proc. Natl. Acad. Sci. USA, 89(18): 8822 (1992)), POMC (Benjannet et al., Proc. Natl. Acad. Sci. USA, 88(9): 3564 (1991)), pro-glucagon (Rouille et al., Proc. Natl. Acad. Sci. USA, 91(8): 3242 (1994)), pro-somatostatin (Xu and Shields, Biochimie 76: 257 (1994)), pro-neurotensin/neuromedin N (proNT/NN; Rovere et al., J. Biol. Chem., 271(19): 11368 (1996)) and pro-melanin concentrating hormone (MCH; Viale et al., J. Biol. Chem., 274(10): 6536 (1999)) C-terminally to KR or RR motifs (see Rouille et al., Front Neuroendocrinol. 16(4):322 (1995)). PC5A is also involved in the processing of proNT/NN and MCH (Barbero et al., J. Biol. Chem., 273(39): 25339 (1998)).

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to a method for decreasing nonprocessed Factor VIII in a culturing medium, comprising,

a) expressing Factor VIII from a first polynucleotide in a host cell, where the endogenous processing enzymes of the host cell are insufficient to convert all of the Factor VIII to its processed isoform;

b) contacting the Factor VIII with an exogenous proprotein convertase; and

c) decreasing the nonprocessed Factor VIII by processing with the proprotein convertase. The exogenous proprotein convertase can be expressed from a second polypeptide in the host cell expressing said Factor VIII, added to said culture medium, or expressed from a polynucleotide sequence in a host cell different from the host cell expressing the Factor VIII.

The Factor VIII of the invention may be processed by cleaving Arginine located at amino acid residue 1648 in SEQ ID NO: 6 (full-length Factor VIII), which corresponds to amino acid residue 1667 in SEQ ID NO: 62, or amino acid residue 754 in SEQ ID NO: 2 (B-domain-deleted Factor VIII), which corresponds to amino acid residue 773 in SEQ ID NO: 60. By the present method, the level of the nonprocessed Factor VIII can be decreased such that the Factor VIII contains more than 75%, 80%, 85%, 90%, 95%, and 100% of the processed Factor VIII after processing by said proprotein convertase.

In one embodiment, the proprotein convertase used in the invention is selected from the group consisting of proprotein convertase subtilisin/kexin type 1, proprotein convertase subtilisin/kexin type 2, proprotein convertase subtilisin/kexin type 3, proprotein convertase subtilisin/kexin type 4, proprotein convertase subtilisin/kexin type 5, proprotein convertase subtilisin/kexin type 6, proprotein convertase subtilisin/kexin type 7, proprotein convertase subtilisin/kexin type 8, proprotein convertase subtilisin/kexin type 9, a yeast Kex 2 and a combination thereof.

In another embodiment, the proprotein convertase for the invention is selected from the group consisting of proprotein convertase subtilisin/kexin type 3, proprotein convertase subtilisin/kexin type 5, proprotein convertase subtilisin/kexin type 7, a yeast Kex 2 and a combination thereof. In a particular embodiment, the proprotein convertase is selected from the group consisting of proprotein convertase subtilisin/kexin type 5, proprotein convertase subtilisin/kexin type 7, and both.

In certain embodiments, the proprotein convertase of the invention comprises an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to proprotein convertase subtilisin/kexin type 5 (SEQ ID NO: 18) wherein the proprotein convertase is capable of cleaving full-length Factor VIII (SEQ ID NO: 6) at amino acid residue 1648 or the B-domain deleted Factor VIII (SEQ ID NO: 2) at amino acid residue 754. In some embodiments, the proprotein convertase of the invention comprises an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to proprotein convertase subtilisin/kexin type 7 (SEQ ID NO: 22) wherein the proprotein convertase is capable of cleaving full-length Factor VIII (SEQ ID NO: 6) at amino acid residue 1648 or the B-domain deleted Factor VIII (SEQ ID NO: 2) at 754.

The Factor VIII in the present invention can be expressed as full-length Factor VIII or a fragment, derivative, analog, or variant thereof. In one embodiment, the Factor VIII is partial or full B-domain deleted Factor VIII. For example, the Factor VIII comprises an amino acid sequence at least 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to SEQ ID NO: 2 or 6 (B-domain-deleted FVIII or full-length Factor VIII, respectively), wherein an activated form of the Factor VIII is capable of restoring clotting activity to FVIII-deficient plasma when activated. In one embodiment, the Factor VIII is fused to an immunoglobulin constant region or a portion thereof, e.g., a neonatal Fc Receptor (FcRn) binding domain, e.g., an Fc fragment. In certain embodiments, the Factor VIII is in a dimer, homodimer, heterodimer, or monomer-dimer hybrid.

In some embodiments, the first polynucleotide encoding Factor VIII and the second polynucleotide encoding a proprotein convertase are located on the same vector or on two different vectors.

In one aspect, the present invention is related to an expression vector comprising a first polynucleotide encoding Factor VIII and a second polynucleotide encoding a proprotein convertase, wherein the Factor VIII is processed by the proprotein convertase when the Factor VIII and the proprotein convertase are expressed in a host cell comprising the vector. Also included in the present invention is a host cell comprising an expression vector comprising a first polynucleotide sequence encoding Factor VIII and a second polynucleotide sequence encoding a proprotein convertase, wherein the Factor VIII is processed by the proprotein convertase.

In another aspect, the present invention is related to a method of producing Factor VIII, comprising

a) culturing the host cell in a culture medium that expresses the Factor VIII and the proprotein convertase, and

b) processing the Factor VIII by the protein convertase.

In some aspects, the present invention includes a method of producing Factor VIII, comprising

a) transfecting the expression vector in a host cell,

b) culturing the host cell in a culture medium to express the Factor VIII and the proprotein convertase, wherein the proprotein convertase processes the Factor VIII.

Also included is an isolated polypeptide comprising Factor VIII produced by the methods or methods of treating a hemophilia using the polypeptide produced by the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

FIG. 1. Schematic Representation of the nonprocessed (upper) and processed rFVIIIBDD-Fc monomer (lower).

FIG. 2. Schematic diagram of Thrombin activation of full-length Factor VIII.

FIG. 3A-B. A. Western blot analysis using anti-Factor VIII A2 (HC) primary antibody. B. Western blot analysis using anti-human IgG-HRP conjugates. The lanes show expression of processed rFVIIIBDD-Fc (˜131 kD for the light chain-Fc fusion and ˜86 kD for the heavy chain) and nonprocessed rFVIIIBDD-Fc (˜217 kD) in HEK293 cells, which are transiently transfected with (1) rFVIIIBDD-Fc expressing construct alone, (2) rFVIIIBDD-Fc and yeast Kex 2 expressing constructs, (3) rFVIIIBDD-Fc and PC7 expressing constructs, (4) rFVIIIBDD-Fc and PACE expressing constructs, and (5) rFVIIIBDD-Fc and PC5 expressing constructs.

FIG. 4A-B. A. SDS-PAGE gel by SYPRO® ruby gel staining. B SDS-PAGE gel by Coomassie gel staining. The lanes show expression of processed rFVIIIBDD-Fc (˜131 kD for the light chain-Fc fusion and ˜86 kD for the heavy chain) and nonprocessed rFVIIIBDD-Fc (˜217 kD) in HEK293 cells, which are transiently transfected with (1) rFVIIIBDD-Fc expressing construct alone, (2) rFVIIIBDD-Fc and yeast Kex 2 expressing constructs, (3) rFVIIIBDD-Fc and PC7 expressing constructs, (4) rFVIIIBDD-Fc and PACE expressing constructs, and (5) rFVIIIBDD-Fc and PC5 expressing constructs.

FIG. 5A-B. A. Reduced SDS-PAGE gel of rFVIIIBDD-Fc. The first lane shows rFVIIIFcBDD-Fc produced after stably cotransfecting a PC5 expressing construct. The second lane shows rFVIIIFcBDD-Fc produced without stably transfecting a processing enzyme expressing construct.

FIG. 6A-B. Western blot analysis of FVIII:Fc produced in media containing Kex2, PC7, PACE, or PC5. FVIII:Fc was detected by anti-FVIII light chain primary antibody (A) or anti-FVIIIA2 (HC) primary antibody (B).

DETAILED DESCRIPTION OF THE INVENTION Definitions

It is to be noted that the term “a” or “an” entity refers to one or more of that entity; for example, “a nucleotide sequence,” is understood to represent one or more nucleotide sequences. As such, the terms “a” (or “an”), “one or more,” and “at least one” can be used interchangeably herein.

The term “polynucleotide” or “nucleotide” is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA) or plasmid DNA (pDNA). In certain embodiments, a polynucleotide comprises a conventional phosphodiester bond or a non-conventional bond (e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term “nucleic acid” refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. By “isolated” nucleic acid or polynucleotide is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment. For example, a recombinant polynucleotide encoding a Factor VIII polypeptide contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) from other polynucleotides in a solution. Isolated RNA molecules include in vivo or in vitro RNA transcripts of polynucleotides of the present invention. Isolated polynucleotides or nucleic acids according to the present invention further include such molecules produced synthetically. In addition, a polynucleotide or a nucleic acid can include regulatory elements such as promoters, enhancers, ribosome binding sites, or transcription termination signals.

As used herein, a “coding region” or “coding sequence” is a portion of polynucleotide which consists of codons translatable into amino acids. Although a “stop codon” (TAG, TGA, or TAA) is typically not translated into an amino acid, it may be considered to be part of a coding region, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, and the like, are not part of a coding region. The boundaries of a coding region are typically determined by a start codon at the 5′ terminus, encoding the amino terminus of the resultant polypeptide, and a translation stop codon at the 3′terminus, encoding the carboxyl terminus of the resulting polypeptide. Two or more coding regions of the present invention can be present in a single polynucleotide construct, e.g., on a single vector, or in separate polynucleotide constructs, e.g., on separate (different) vectors. It follows, then, that a single vector can contain just a single coding region, or comprise two or more coding regions, e.g., a single vector can separately encode a binding domain-A and a binding domain-B as described below. In addition, a vector, polynucleotide, or nucleic acid of the invention can encode heterologous coding regions, either fused or unfused to a nucleic acid encoding a binding domain of the invention. Heterologous coding regions include without limitation specialized elements or motifs, such as a secretory signal peptide or a heterologous functional domain.

Certain proteins secreted by mammalian cells are associated with a secretory signal peptide which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated. Those of ordinary skill in the art are aware that signal peptides are generally fused to the N-terminus of the polypeptide, and are cleaved from the complete or “full-length” polypeptide to produce a secreted or “mature” form of the polypeptide. In certain embodiments, a native signal peptide, e.g., an immunoglobulin heavy chain or light chain signal peptide is used, or a functional derivative of that sequence that retains the ability to direct the secretion of the polypeptide that is operably associated with it. Alternatively, a heterologous mammalian signal peptide, e.g., a human tissue plasminogen activator (TPA) or mouse β-glucuronidase signal peptide, or a functional derivative thereof, can be used.

The term “downstream” refers to a nucleotide sequence that is located 3′ to a reference nucleotide sequence. In certain embodiments, downstream nucleotide sequences relate to sequences that follow the starting point of transcription. For example, the translation initiation codon of a gene is located downstream of the start site of transcription.

The term “upstream” refers to a nucleotide sequence that is located 5′ to a reference nucleotide sequence. In certain embodiments, upstream nucleotide sequences relate to sequences that are located on the 5′ side of a coding region or starting point of transcription. For example, most promoters are located upstream of the start site of transcription.

As used herein, the term “regulatory region” refers to nucleotide sequences located upstream (5′ non-coding sequences), within, or downstream (3′ non-coding sequences) of a coding region, and which influence the transcription, RNA processing, stability, or translation of the associated coding region. Regulatory regions may include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem-loop structures. If a coding region is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3′ to the coding sequence.

A polynucleotide which encodes a gene product, e.g., a polypeptide, can include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. In an operable association a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory regions in such a way as to place expression of the gene product under the influence or control of the regulatory region(s). For example, a coding region and a promoter are “operably associated” if induction of promoter function results in the transcription of mRNA encoding the gene product encoded by the coding region, and if the nature of the linkage between the promoter and the coding region does not interfere with the ability of the promoter to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can also be operably associated with a coding region to direct gene product expression.

A variety of transcription control regions are known to those skilled in the art. These include, without limitation, transcription control regions which function in vertebrate cells, such as, but not limited to, promoter and enhancer segments from cytomegaloviruses (the immediate early promoter, in conjunction with intron-A), simian virus 40 (the early promoter), and retroviruses (such as Rous sarcoma virus). Other transcription control regions include those derived from vertebrate genes such as actin, heat shock protein, bovine growth hormone and rabbit β-globin, as well as other sequences capable of controlling gene expression in eukaryotic cells. Additional suitable transcription control regions include tissue-specific promoters and enhancers as well as lymphokine-inducible promoters (e.g., promoters inducible by interferons or interleukins).

Similarly, a variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from picornaviruses (particularly an internal ribosome entry site, or IRES, also referred to as a CITE sequence).

The term “expression” as used herein refers to a process by which a polynucleotide produces a gene product, for example, an RNA or a polypeptide. It includes without limitation transcription of the polynucleotide into messenger RNA (mRNA), transfer RNA (tRNA), small hairpin RNA (shRNA), small interfering RNA (siRNA) or any other RNA product, and the translation of an mRNA into a polypeptide. Expression produces a “gene product.” As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation, glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage.

A “vector” refers to any vehicle for the cloning of and/or transfer of a nucleic acid into a host cell. A vector may be a replicon to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. A “replicon” refers to any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of replication in vivo, i.e., capable of replication under its own control. The term “vector” includes both viral and nonviral vehicles for introducing the nucleic acid into a cell in vitro, ex vivo or in vivo. A large number of vectors are known and used in the art including, for example, plasmids, modified eukaryotic viruses, or modified bacterial viruses. Insertion of a polynucleotide into a suitable vector can be accomplished by ligating the appropriate polynucleotide fragments into a chosen vector that has complementary cohesive termini.

Vectors may be engineered to encode selectable markers or reporters that provide for the selection or identification of cells that have incorporated the vector. Expression of selectable markers or reporters allows identification and/or selection of host cells that incorporate and express other coding regions contained on the vector. Examples of selectable marker genes known and used in the art include: genes providing resistance to ampicillin, streptomycin, gentamycin, kanamycin, hygromycin, bialaphos herbicide, sulfonamide, and the like; and genes that are used as phenotypic markers, i.e., anthocyanin regulatory genes, isopentanyl transferase gene, and the like. Examples of reporters known and used in the art include: luciferase (Luc), green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), -galactosidase (LacZ), -glucuronidase (Gus), and the like. Selectable markers may also be considered to be reporters.

The term “plasmid” refers to an extra-chromosomal element often carrying a gene that is not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear, circular, or supercoiled, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3′ untranslated sequence into a cell.

Eukaryotic viral vectors that can be used include, but are not limited to, adenovirus vectors, retrovirus vectors, adeno-associated virus vectors, poxvirus, e.g., vaccinia virus vectors, baculovirus vectors, or herpesvirus vectors. Non-viral vectors include plasmids, liposomes, electrically charged lipids (cytofectins), DNA-protein complexes, and biopolymers.

A “cloning vector” refers to a “replicon,” which is a unit length of a nucleic acid that replicates sequentially and which comprises an origin of replication, such as a plasmid, phage or cosmid, to which another nucleic acid segment may be attached so as to bring about the replication of the attached segment. Certain cloning vectors are capable of replication in one cell type, e.g., bacteria and expression in another, e.g., eukaryotic cells. Cloning vectors typically comprise one or more sequences that can be used for selection of cells comprising the vector and/or one or more multiple cloning sites for insertion of nucleic acid sequences of interest.

The term “expression vector” refers to a vehicle designed to enable the expression of an inserted nucleic acid sequence following insertion into a host cell. The inserted nucleic acid sequence is placed in operable association with regulatory regions as described above.

Vectors are introduced into host cells by methods well known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter.

“Culture,” “to culture” and “culturing,” as used herein, means to incubate cells under in vitro conditions that allow for cell growth or division or to maintain cells in a living state. “Cultured cells,” as used herein, means cells that are propagated in vitro.

As used herein, the term “polypeptide” is intended to encompass a singular “polypeptide” as well as plural “polypeptides,” and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein,” “amino acid chain,” or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of “polypeptide,” and the term “polypeptide” can be used instead of, or interchangeably with any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. A polypeptide can be derived from a natural biological source or produced recombinant technology, but is not necessarily translated from a designated nucleic acid sequence. It can be generated in any manner, including by chemical synthesis.

An “isolated” polypeptide or a fragment, variant, or derivative thereof refers to a polypeptide that is not in its natural milieu. No particular level of purification is required. For example, an isolated polypeptide can simply be removed from its native or natural environment. Recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention, as are native or recombinant polypeptides which have been separated, fractionated, or partially or substantially purified by any suitable technique.

Also included in the present invention are fragments or variants of polypeptides, and any combination thereof. The term “fragment” or “variant” when referring to polypeptide binding domains or binding molecules of the present invention include any polypeptides which retain at least some of the properties (e.g., FcRn binding affinity for an FcRn binding domain or Fc variant, coagulation activity for an FVIII variant, or Factor VIII cleaving activity for a protein convertase variant) of the reference polypeptide. Fragments of polypeptides include proteolytic fragments, as well as deletion fragments, in addition to specific antibody fragments discussed elsewhere herein. Variants of polypeptide binding domains or binding molecules of the present invention include fragments as described above, and also polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, or insertions. Variants can be naturally or non-naturally occurring. Non-naturally occurring variants can be produced using art-known mutagenesis techniques. Variant polypeptides can comprise conservative or non-conservative amino acid substitutions, deletions or additions.

A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the substitution is considered to be conservative. In another embodiment, a string of amino acids can be conservatively replaced with a structurally similar string that differs in order and/or composition of side chain family members.

As known in the art, “sequence identity” between two polypeptides is determined by comparing the amino acid sequence of one polypeptide to the sequence of a second polypeptide. When discussed herein, whether any particular polypeptide is at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% identical to another polypeptide can be determined using methods and computer programs/software known in the art such as, but not limited to, the BESTFIT program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711). BESTFIT uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences. When using BESTFIT or any other sequence alignment program to determine whether a particular sequence is, for example, 95% identical to a reference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full-length of the reference polypeptide sequence and that gaps in homology of up to 5% of the total number of amino acids in the reference sequence are allowed.

A “fusion” or chimeric protein comprises a first amino acid sequence linked to a second amino acid sequence with which it is not naturally linked in nature. The amino acid sequences which normally exist in separate proteins can be brought together in the fusion polypeptide, or the amino acid sequences which normally exist in the same protein can be placed in a new arrangement in the fusion polypeptide, e.g., fusion of a Factor VIII domain of the invention with an immunoglobulin Fc domain. A fusion protein is created, for example, by chemical synthesis, or by creating and translating a polynucleotide in which the peptide regions are encoded in the desired relationship.

The term “heterologous” as applied to a polynucleotide or a polypeptide, means that the polynucleotide or polypeptide is derived from a distinct entity from that of the entity to which it is being compared. For instance, a heterologous polynucleotide or antigen can be derived from a different species, different cell type of an individual, or the same or different type of cell of distinct individuals.

As used herein the term “associate with” refers a covalent or non-covalent bond formed between two sulfur atoms. The amino acid cysteine comprises a thiol group that can form a disulfide bond or bridge with a thiol group on a second cysteine residue. In most naturally occurring IgG molecules, the CH1 and CL regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229, EU numbering system).

Methods of Production

The present invention is directed to a method of producing processed Factor VIII by co-expressing Factor VIII and a proprotein convertase or adding a proprotein convertase protein to the Factor VIII expressing host cell or the medium containing the host cell so that the proprotein convertase cleaves Factor VIII at amino acid 1648 (full-length Factor VIII or SEQ ID NO: 6), which corresponds to amino acid residue 1667 of SEQ ID NO: 62, or the corresponding amino acid residue in other variants (e.g., amino acid 754 in the S743/Q1638 B-domain deleted Factor VIII or SEQ ID NO: 2 or amino acid 773 in SEQ ID NO: 60), and thereby generates a Factor VIII heavy chain and a Factor VIII light chain—processed Factor VIII. In the culture or host cell, the present method therefore increases the level of processed Factor VIII by co-expressing Factor VIII and a proprotein convertase in the same host cell or different host cells or adding a proprotein convertase protein to the Factor VIII expressing host cell or the medium containing the host cell, compared to the level of processed Factor VIII without co-expressing the two proteins together. Alternatively, the present method decreases nonprocessed Factor VIII in a culture medium or host cell compared to the level of nonprocessed Factor VIII in the host cell or culture medium without the proprotein convertase. In some embodiments, the present invention is drawn to a method of cleaving Factor VIII by co-expressing Factor VIII and a proprotein convertase, wherein the proprotein convertase cleaves Factor VIII at amino acid 1648 (full-length FVIII or SEQ ID NO: 6) or the corresponding amino acid residue in other variants (e.g., amino acid 754 in the S743/Q1638 B-domain deleted Factor VIII or SEQ ID NO: 2), and generates a FVIII heavy chain and a FVIII light chain.

Factor VIII can be expressed as a single protein in a cell and processed by intracellular processing enzymes to a heavy chain and a light chain. When Factor VIII is expressed in vitro in an excess amount, the intracellular processing enzymes may not be capable of processing all Factor VIII in the host cell. Thus, the present invention provides that a proprotein convertase co-expressed with Factor VIII can cleave the nonprocessed Factor VIII when endogenous processing enzymes are insufficient to process all Factor VIII produced in the host cell.

The term “processed Factor VIII” as used herein means Factor VIII that has been cleaved right after Arginine at amino acid 1648 (in full-length Factor VIII or SEQ ID NO: 6), amino acid 754 (in the S743/Q1638 B-domain deleted Factor VIII or SEQ ID NO: 2), or the corresponding Arginine residue (in other variants). In one embodiment, a processed Factor VIII comprises a heavy chain and a light chain, which are linked or associated by a metal ion-mediated non-covalent bond. The term “nonprocessed Factor VIII” therefore means Factor VIII that has not been cleaved right after Arginine at amino acid 1648 (in full-length Factor VIII or SEQ ID NO: 6), amino acid 754 (in the S743/Q1638 B-domain-deleted Factor VIII or SEQ ID NO: 2), or the corresponding Arginine residue (in other variants). The term “nonprocessed Factor VIII” is interchangeably used herein with “non-processed Factor VIII,” “non processed Factor VIII,” “unprocessed Factor VIII,” “npFactor VIII,” or “npFVIII.”

Processed Factor VIII can further be cleaved by thrombin and then activated as Factor VIIIa, serving as a cofactor for activated Factor IX (FIXa). The activated FIX together with activated FVIII forms a Xase complex and converts Factor X to activated Factor X (FXa). For activation, Factor VIII is cleaved by thrombin after three Arginine residues, at amino acids 372, 740, and 1689 (corresponding to amino acids 372, 740, and 795 in the B-domain deleted FVIII sequence), the cleavage generating Factor VIIIa having the 50 kDa A1, 43 kDa A2, and 73 kDa A3-C1-C2 chains.

“Factor VIII,” as used herein, means functional factor VIII polypeptide in its normal role in coagulation, unless otherwise specified. Thus, the term Factor VIII includes variant polypeptides that are functional. In one embodiment, the Factor VIII protein is the human, porcine, canine, rat, or murine Factor VIII protein. In addition, comparisons between factor VIII from humans and other species have identified conserved residues that are likely to be required for function (Cameron et al., Thromb. Haemost. 79:317-22 (1998); U.S. Pat. No. 6,251,632), incorporated herein by reference in its entirety. In another embodiment, the Factor VIII protein is a chimeric polypeptide, for example, but not limited to, a fusion protein comprising a fully or partially B-domain deleted Factor VIII and at least a portion of an immunoglobulin constant region, e.g., an Fc domain.

The Factor VIII polypeptide and polynucleotide sequences are known, as are many functional fragments, mutants and modified versions. Examples of human factor VIII sequences are shown as subsequences in SEQ ID NO: 2 or 6. Factor VIII polypeptides include full-length Factor VIII, full-length Factor VIII minus Met at the N-terminus, mature Factor VIII (minus the signal sequence), mature Factor VIII with an additional Met at the N-terminus, and/or Factor VIII with a full or partial deletion of the B domain. In certain embodiments, Factor VIII variants include B domain deletions, whether partial or full deletions.

The human factor VIII gene was isolated and expressed in mammalian cells (Toole, J. J., et al., Nature 312:342-347 (1984); Gitschier, J., et al., Nature 312:326-330 (1984); Wood, W. L, et al, Nature 312:330-337 (1984); Vehar, G. A., et al, Nature 312:337-342 (1984); WO 87/04187; WO 88/08035; WO 88/03558; and U.S. Pat. No. 4,757,006), each of which is incorporated herein by reference in its entirety. The Factor VIII amino acid sequence was deduced from cDNA as shown in U.S. Pat. No. 4,965,199, which is incorporated herein by reference in its entirety. In addition, partially or fully B-domain deleted Factor VIII is shown in U.S. Pat. Nos. 4,994,371 and 4,868,112, each of which is incorporated herein by reference in its entirety. In some embodiments, the human Factor VIII B-domain is replaced with the human Factor V B-domain as shown in U.S. Pat. No. 5,004,803, incorporated herein by reference in its entirety. The cDNA sequence encoding human Factor VIII and predicted amino acid sequence are shown in SEQ ID NOs:1 and 2, respectively, of US Application Publ. No. 2005/0100990, incorporated herein by reference in its entirety.

The porcine factor VIII sequence is published in Toole, J. J., et al., Proc. Natl. Acad. Sci. USA 83:5939-5942 (1986), which is incorporated herein by reference in its entirety. And the complete porcine cDNA sequence obtained from PCR amplification of Factor VIII sequences from a pig spleen cDNA library has been reported in Healey, J. F., et al., Blood 88:4209-4214 (1996), incorporated herein by reference in its entirety. Hybrid human/porcine Factor VIII having substitutions of all domains, all subunits, and specific amino acid sequences were disclosed in U.S. Pat. No. 5,364,771 by Lollar and Runge, and in WO 93/20093, incorporated herein by reference in its entirety. More recently, the nucleotide and corresponding amino acid sequences of the A1 and A2 domains of porcine factor VIII and a chimeric factor VIII with porcine A1 and/or A2 domains substituted for the corresponding human domains were reported in WO 94/11503, incorporated herein by reference in its entirety. U.S. Pat. No. 5,859,204, Lollar, J. S., also discloses the porcine cDNA and deduced amino acid sequences. U.S. Pat. No. 6,458,563, incorporated herein by reference in its entirety assigned to Emory discloses a B-domain-deleted porcine Factor VIII.

U.S. Pat. No. 5,859,204, Lollar, J. S., incorporated herein by reference in its entirety, reports functional mutants of Factor VIII having reduced antigenicity and reduced immunoreactivity. U.S. Pat. No. 6,376,463, Lollar, J. S., incorporated herein by reference in its entirety, also reports mutants of Factor VIII having reduced immunoreactivity. US Appl. Publ. No. 2005/0100990, Saenko et al., incorporated herein by reference in its entirety, reports functional mutations in the A2 domain of Factor VIII.

In one embodiment, the Factor VIII (or Factor VIII portion of a chimeric polypeptide) may be at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a Factor VIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO: 6 (without a signal sequence) or a Factor VIII amino acid sequence of amino acids −19 to 1438 of SEQ ID NO: 2 or amino acids −19 to 2332 of SEQ ID NO: 6 (with a signal sequence), wherein the Factor VIII has a clotting activity, e.g., activates Factor IX as a cofactor to convert Factor X to activated Factor X. The Factor VIII (or Factor VIII portion of a chimeric polypeptide) may be identical to a Factor VIII amino acid sequence of amino acids 1 to 1438 of SEQ ID NO: 2 or amino acids 1 to 2332 of SEQ ID NO:6 (without a signal sequence). The Factor VIII may further comprise a signal sequence.

“B-domain” of Factor VIII, as used herein, is the same as the B-domain known in the art that is defined by internal amino acid sequence identity and sites of proteolytic cleavage, e.g., residues Ser741-Arg1648 of full-length human Factor VIII. The other human Factor VIII domains are defined by the following amino acid residues: A1, residues A1a1-Arg372; A2, residues Ser373-Arg740; A3, residues Ser1690-Asn2019; C1, residues Lys2020-Asn2172; C2, residues Ser2173-Tyr2332. The A3-C1-C2 sequence includes residues Ser1690-Tyr2332. The remaining sequence, residues Glu1649-Arg1689, is usually referred to as the a3 acidic region. The locations of the boundaries for all of the domains, including the B-domains, for porcine, mouse and canine Factor VIII are also known in the art. In one embodiment, the B domain of Factor VIII is deleted (“B-domain-deleted factor VIII” or “BDD FVIII”). An example of a BDD FVIII is REFACTO® (recombinant BDD FVIII), which has the same sequence as the Factor VIII portion of the sequence in Table 2A(i) (amino acids −19 to 1438 or 1 to 1438 of SEQ ID NO:2).

A “B-domain-deleted Factor VIII” may have the full or partial deletions disclosed in U.S. Pat. Nos. 6,316,226, 6,346,513, 7,041,635, 5,789,203, 6,060,447, 5,595,886, 6,228,620, 5,972,885, 6,048,720, 5,543,502, 5,610,278, 5,171,844, 5,112,950, 4,868,112, and 6,458,563, each of which is incorporated herein by reference in its entirety. In some embodiments, a B-domain-deleted Factor VIII sequence of the present invention comprises any one of the deletions disclosed at col. 4, line 4 to col. 5, line 28 and examples 1-5 of U.S. Pat. No. 6,316,226 (also in U.S. Pat. No. 6,346,513). In another embodiment, a B-domain deleted Factor VIII is the S743/Q1638 B-domain deleted Factor VIII (SQ version Factor VIII) (e.g., Factor VIII having a deletion from amino acid 744 to amino acid 1637, e.g., Factor VIII having amino acids 1-743 and amino acids 1638-2332 of SEQ ID NO: 6, i.e., SEQ ID NO: 2). In some embodiments, a B-domain-deleted Factor VIII of the present invention has a deletion disclosed at col. 2, lines 26-51 and examples 5-8 of U.S. Pat. No. 5,789,203 (also U.S. Pat. No. 6,060,447, U.S. Pat. No. 5,595,886, and U.S. Pat. No. 6,228,620). In some embodiments, a B-domain-deleted Factor VIII has a deletion described in col. 1, lines 25 to col. 2, line 40 of U.S. Pat. No. 5,972,885; col. 6, lines 1-22 and example 1 of U.S. Pat. No. 6,048,720; col. 2, lines 17-46 of U.S. Pat. No. 5,543,502; col. 4, line 22 to col. 5, line 36 of U.S. Pat. No. 5,171,844; col. 2, lines 55-68, FIG. 2, and example 1 of U.S. Pat. No. 5,112,950; col. 2, line 2 to col. 19, line 21 and table 2 of U.S. Pat. No. 4,868,112; col. 2, line 1 to col. 3, line 19, col. 3, line 40 to col. 4, line 67, col. 7, line 43 to col. 8, line 26, and col. 11, line 5 to col. 13, line 39 of U.S. Pat. No. 7,041,635; or col. 4, lines 25-53, of U.S. Pat. No. 6,458,563. In some embodiments, a B-domain-deleted Factor VIII has a deletion of most of the B domain, but still contains amino-terminal sequences of the B domain that are essential for in vivo proteolytic processing of the primary translation product into two polypeptide chain, as disclosed in WO 91/09122, which is incorporated herein by reference in its entirety. In some embodiments, a B-domain-deleted Factor VIII is constructed with a deletion of amino acids 747-1638, i.e., virtually a complete deletion of the B domain. Hoeben R. C., et al. J. Biol. Chem. 265 (13): 7318-7323 (1990), incorporated herein by reference in its entirety. A B-domain-deleted Factor VIII may also contain a deletion of amino acids 771-1666 or amino acids 868-1562 of Factor VIII. Meulien P., et al. Protein Eng. 2(4): 301-6 (1988), incorporated herein by reference in its entirety. Additional B domain deletions that are part of the invention include: deletion of amino acids 982 through 1562 or 760 through 1639 (Toole et al., Proc. Natl. Acad. Sci. U.S.A. (1986) 83, 5939-5942)), 797 through 1562 (Eaton, et al. Biochemistry (1986) 25:8343-8347)), 741 through 1646 (Kaufman (PCT published application No. WO 87/04187)), 747-1560 (Sarver, et al., DNA (1987) 6:553-564)), 741 though 1648 (Pasek (PCT application No. 88/00831)), or 816 through 1598 or 741 through 1648 (Lagner (Behring Inst. Mitt. (1988) No 82:16-25, EP 295597)), each of which is incorporated herein by reference in its entirety. Each of the foregoing deletions may be made in any Factor VIII sequence.

In one embodiment, Factor VIII processed using the present methods is in a form of a chimeric polypeptide. “Chimeric polypeptide,” as used herein, means a polypeptide that includes within it at least two polypeptides (or subsequences or peptides) from different sources, e.g., a heterologous polypeptide. Chimeric polypeptides may include two, three, four, five, six, seven, or more polypeptides from different sources, such as different genes, different cDNAs, or different animal or other species. Chimeric polypeptides may include one or more linkers joining the different subsequences. Thus, the subsequences may be joined directly or indirectly, via linkers, or both, within a single chimeric polypeptide. Chimeric polypeptides may include additional peptides such as signal sequences and sequences such as 6His and FLAG that aid in protein purification or detection. In addition, chimeric polypeptides may have amino acid or peptide additions to the N- and/or C-termini. Exemplary chimeric polypeptides of the invention are Factor VIII-Fc chimeric polypeptides such as SEQ ID NO: 60 or 62, with or without their signal sequences and the chimeric Fc polypeptide of SEQ ID NO:4.

The Factor VIII polypeptide fused to an Fc domain may comprise a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Factor VIII and Fc amino acid sequence of SEQ ID NO: 60, wherein the Factor VIII portion has the Factor VIII clotting activity, e.g., activating Factor IX as a cofactor to convert Factor X to activated Factor X (FXa) and the Fc domain portion has the FcRn binding affinity.

In some embodiments, the Factor VIII polypeptide fused to an Fc domain may comprise a sequence at least 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Factor VIII and Fc amino acid sequence of SEQ ID NO: 62, wherein the Factor VIII portion has the Factor VIII clotting activity, e.g., activating Factor IX as a cofactor to convert Factor X to activated Factor X (FXa) and the Fc domain portion has the FcRn binding affinity. The Factor VIII polypeptide fused to an Fc domain may further comprise a signal sequence.

In certain embodiments, Factor VIII used in this invention is conjugated. As used herein, a conjugate refers to any two or more entities bound to one another by any physicochemical means, including, but not limited to, hydrophobic interaction, covalent interaction, hydrogen bond interaction, ionic interaction, or any combination thereof. Thus, in one embodiment, a conjugate of the invention refers to any two or more entities bound to one another by covalent interaction. For example, in one embodiment, a conjugate is a fusion protein. In another embodiment, a conjugate of the invention refers to any two or more entities bound to one another by noncovalent interaction.

In one embodiment, Factor VIII of this invention is fused to an antibody or a fragment thereof, e.g., at least a portion of an immunoglobulin constant region. Immunoglobulins are comprised of four polypeptide chains, two heavy chains and two light chains, which associate via disulfide bonds to form tetramers. Each chain is further comprised of one variable region and one or more constant regions. The variable regions mediate antigen recognition and binding, while the constant regions, particularly the heavy chain constant regions, mediate a variety of effector functions, e.g., complement binding and Fc receptor binding (see, e.g., U.S. Pat. Nos. 6,086,875; 5,624,821; 5,116,964).

The constant region is further comprised of domains denoted CH (constant heavy) domains (CH1, C1-12, etc.). Depending on the isotype, IgG, IgM, IgA IgD, or IgE) the constant region can be comprised of three or four CH domains. Some isotypes (e.g. IgG) constant regions also contain a hinge region. See Janeway et al. 2001, Immunobiology, Garland Publishing, N.Y., N.Y.

The creation of chimeric proteins comprised of immunoglobulin constant regions linked to a protein of interest, or fragment thereof, has been described (see, e.g., U.S. Pat. Nos. 5,480,981 and 5,808,029; Gascoigne et al. 1987, Proc. Natl. Acad. Sci. USA 84:2936; Capon et al. 1989, Nature 337:525; Traunecker et al. 1989, Nature 339:68; Zettmeissl et al. 1990, DNA Cell Biol. USA 9:347; Byrn et al. 1990, Nature 344:667; Watson et al. 1990, J. Cell. Biol. 110:2221; Watson et al. 1991, Nature 349:164; Aruffo et al. 1990, Cell 61:1303; Linsley et al. 1991, J. Exp. Med. 173:721; Linsley et al. 1991, J. Exp. Med. 174:561; Stamenkovic et al., 1991, Cell 66:1133; Ashkenazi et al. 1991, Proc. Natl. Acad. Sci. USA 88:10535; Lesslauer et al. 1991, Eur. J. Immunol. 27:2883; Peppel et al. 1991, J. Exp. Med. 174:1483; Bennett et al. 1991, J. Biol. Chem. 266:23060; Kurschner et al. 1992, J. Biol. Chem. 267:9354; Chalupny et al. 1992, Proc. Natl. Acad. Sci. USA 89:10360; Ridgway and Gorman, 1991, J. Cell. Biol. 115, Abstract No. 1448; Zheng et al. 1995, J. Immun. 154:5590). These molecules usually possess both the biological activity associated with the linked molecule of interest as well as the effector function, and possibly some other desired characteristic associated with the immunoglobulin constant region (e.g., biological stability, cellular secretion).

The Fc portion of an immunoglobulin constant region, depending on the immunoglobulin isotype can include the CH2, CH3, and CH4 domains, as well as the hinge region. Chimeric proteins comprising an Fc portion of an immunoglobulin bestow several desirable properties on a chimeric protein including increased stability, increased serum half-life (see Capon et al., 1989, Nature 337:525) as well as binding to Fc receptors such as the neonatal Fc receptor (FcRn) (U.S. Pat. Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1), which are incorporated herein by reference in their entireties.

FcRn is active in adult epithelial tissues and expressed in the lumen of the intestines, pulmonary airways, nasal surfaces, vaginal surfaces, colon and rectal surfaces (U.S. Pat. No. 6,485,726). Fusion proteins comprising FcRn binding partners (e.g. IgG, Fc fragments) can be effectively shuttled across epithelial barriers by FcRn, thus providing a non-invasive means to systemically administer a desired therapeutic molecule. Additionally, fusion proteins comprising an FcRn binding partner are endocytosed by cells expressing the FcRn. But instead of being marked for degradation, these fusion proteins are recycled out into circulation again, thus increasing the in vivo half-life of these proteins.

Portions of immunoglobulin (e.g., IgG) constant regions, e.g., FcRn binding partners typically associate, via disulfide bonds and other noncovalent interactions, with one another to form dimers. In one embodiment, Factor VIII is fused to an Fc domain as a monomer. In another embodiment, Factor VIII is fused to an Fc domain as a dimer, homodimer or heterodimer. In some embodiments, the Factor VIII-Fc fusion is associated with a second polypeptide chain comprising, consisting essentially of, or consisting of at least a portion of an immunoglobulin constant region comprising an FcRn binding domain (“monomer-dimer hybrid”).

“Hybrid” polypeptides and proteins, as used herein, means a combination of a first polypeptide chain, e.g., Factor VIII-Fc fusion, with a second polypeptide chain. In one embodiment, the first polypeptide and the second polypeptide in a hybrid are associated with each other via protein-protein interactions, such as charge-charge or hydrophobic interactions. In another embodiment, the first polypeptide and the second polypeptide in a hybrid are associated with each other via disulfide or other covalent bond(s). Hybrids are described in US 2004/101740 and US 2006/074199, each of which is incorporated herein by reference in its entirety. The second polypeptide may be an identical copy of the first polypeptide or a non-identical polypeptide. In one embodiment, the first polypeptide is a Factor VIII-Fc fusion protein, and the second polypeptide is a polypeptide comprising, consisting essentially of, or consisting of an FcRn binding domain, wherein the first polypeptide and the second polypeptide are associated. In another embodiment, the first polypeptide comprises FVIII-Fc, and the second polypeptide comprises FVIII-Fc, making the hybrid a homodimer.

“Fc,” as used herein, comprises functional neonatal Fc receptor (FcRn) binding partners, unless otherwise specified. An FcRn binding partner is any molecule that can be specifically bound by the FcRn receptor with consequent active transport by the FcRn receptor of the FcRn binding partner. Thus, the term Fc includes any variants of IgG Fc that are functional. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994, Nature 372:379, incorporated herein by reference in its entirety). The major contact area of the Fc with the FcRn is near the junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig heavy chain. The FcRn binding partners include whole IgG, the Fc fragment of IgG, and other fragments of IgG that include the complete binding region of FcRn. The major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290-291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428, and 433-436 of the CH3 domain. References made to amino acid numbering of immunoglobulins or immunoglobulin fragments, or regions, are all based on Kabat et al. 1991, Sequences of Proteins of Immunological Interest, U. S. Department of Public Health, Bethesda; MD, incorporated herein by reference in its entirety. (The FcRn receptor has been isolated from several mammalian species including humans. The sequences of the human FcRn, rat FcRn, and mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180: 2377), incorporated herein by reference in its entirety.) An Fc may comprise the CH2 and CH3 domains of an immunoglobulin with or without the hinge region of the immunoglobulin. Exemplary Fc variants are provided in WO 2004/101740 and WO 2006/074199, incorporated herein by reference in their entireties.

Fc (or Fc portion of a chimeric polypeptide) as used herein may contain one or more mutations or combinations of mutations. In one embodiment, a mutation in Fc confers increased half-life; for example, the mutation may include M252Y, S254T, T256E, and/or combinations thereof as disclosed in Oganesyan et al., Mol. Immunol. 46:1750 (2009), which is incorporated herein by reference in its entirety; H433K, N434F, and/or combinations thereof, as disclosed in Vaccaro et al., Nat. Biotechnol. 23:1283 (2005), which is incorporated herein by reference in its entirety; the mutants disclosed at pages 1-2, paragraph [0012], and Examples 9 and 10 of US 2009/0264627 A1, which is incorporated herein by reference in its entirety; or the mutants disclosed at page 2, paragraphs [0014] to [0021] of US 20090163699 A1, which is incorporated herein by reference in its entirety. In another embodiment, a mutation in Fc preserves or enhances binding to the FcRn; for example, such mutations may include the mutants disclosed in paragraphs [148]-[150] of U.S. Pat. No. 7,404,956 BI, which is incorporated herein by reference in its entirety.

In one embodiment, the FcRn binding domain is a polypeptide including the sequence PKNSSMISNTP (SEQ ID NO: 27) and optionally further including a sequence selected from HQSLGTQ (SEQ ID NO: 28), HQNLSDGK (SEQ ID NO: 29), HQNISDGK (SEQ ID NO: 30), or VISSHLGQ (SEQ ID NO: 31), which are disclosed in U.S. Pat. No. 5,739,277, incorporated herein by reference in its entirety.

In another embodiment, the FcRn binding domain, e.g., Fc (or Fc portion of a chimeric polypeptide), may be at least 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the Fc amino acid sequence of SEQ ID NO: 4.

In some embodiments, Factor VIII may be PEGylated. PEGylated Factor VIII can refer to a conjugate formed between Factor VIII and at least one polyethylene glycol (PEG) molecule. PEG is commercially available in a large variety of molecular weights and average molecular weight ranges. Typical examples of PEG average molecular weight ranges include, but are not limited to, 200, 300, 400, 600, 1000, 1300-1600, 1450, 2000, 3000, 3000-3750, 3350, 3000-7000, 3500-4500, 5000-7000, 7000-9000, 8000, 10000, 8500-11500, 16000-24000, 35000, and 40000. These average molecular weights are provided merely as examples and are not meant to be limiting in any way.

In one embodiment, the Factor VIII portion of the invention may be PEGylated to include mono- or poly- (e.g., 2-4) PEG moieties. PEGylation may be carried out by any of the PEGylation reactions known in the art. Methods for preparing a PEGylated protein product will generally include (a) reacting a polypeptide with polyethylene glycol (such as a reactive ester or aldehyde derivative of PEG) under conditions whereby the peptide of the invention becomes attached to one or more PEG groups; and (b) obtaining the reaction product(s). In general, the optimal reaction conditions for the reactions will be determined case by case based on known parameters and the desired result.

There are a number of PEG attachment methods available to those skilled in the art, for example, EP 0 401 384; Malik F et al. (1992) Exp Hematol. 20:1028-35; Francis (1992) Focus on Growth Factors 3(2):4-10; EP 0 154 316; EP 0 401 384; WO 92/16221; and WO 95/34326. As a non-limiting example, Factor VIII variants can contain cysteine substitutions at the surface-exposed sites, and the cysteines can be further conjugated to PEG polymer. Mei et al., Blood, Mar. 1, 2010, DOI 10.1182/blood-2009-11-254755, which is incorporated herein by reference in its entirety.

The step of PEGylation as described for the proteins of the invention may be carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule. Thus, protein products according to the present invention include PEGylated proteins wherein the PEG group(s) is (are) attached via acyl or alkyl groups. Such products may be mono-PEGylated or poly-PEGylated (for example, those containing 2-6 or 2-5 PEG groups). The PEG groups are generally attached to the protein at the α.- or ε-amino groups of amino acids, but it is also contemplated that the PEG groups can be attached to any amino group attached to the protein that is sufficiently reactive to become attached to a PEG group under suitable reaction conditions.

PEGylation by acylation generally involves reacting an active ester derivative of polyethylene glycol with a peptide of the invention. For acylation reactions, the polymer(s) selected typically have a single reactive ester group. Any known or subsequently discovered reactive PEG molecule may be used to carry out the PEGylation reaction. An example of a suitable, activated PEG ester is PEG esterified to N-hydroxysuccinimide (NHS). As used herein, acylation is contemplated to include, without limitation, the following types of linkages between the therapeutic protein and a polymer such as PEG: amide, carbamate, urethane, and the like. See, for example, Chamow S M et al (1994) Bioconjug Chem. 5:133-40. Reaction conditions may be selected from any of those known in the PEGylation art or those subsequently developed, but should avoid conditions such as temperature, solvent, and pH that would inactivate the polypeptide to be modified.

PEGylation by acylation will generally result in a poly-PEGylated protein. The connecting linkage may be an amide. The resulting product may be substantially only (e.g., >95%) mono-, di- or tri-PEGylated. However, some species with higher degrees of PEGylation may be formed in amounts depending on the specific reaction conditions used. If desired, more purified PEGylated species may be separated from the mixture (particularly unreacted species) by standard purification techniques, including among others, dialysis, salting-out, ultrafiltration, ion-exchange chromatography, gel filtration chromatography and electrophoresis.

PEGylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with a polypeptide in the presence of a reducing agent. For the reductive alkylation reaction, the polymer(s) selected should have a single reactive aldehyde group. An exemplary reactive PEG aldehyde is polyethylene glycol propionaldehyde, which is water stable, or mono C1-C10 alkoxy or aryloxy derivatives thereof. See, for example, U.S. Pat. No. 5,252,714.

The Factor VIII portion of the present invention can also be glycoPEGylated. “GlycoPEGylated” Factor VIII can be produced by conjugating glycosylated or non-glycosylated Factor VIII with a glycosyl moiety that includes a polymer moiety, e.g., poly(ethylene glycol), alkyl derivative (e.g., m-PEG), or reactive derivative (e.g., H2N-PEG, HOOC-PEG), within its structure. In one embodiment, the PEG moiety is attached to the glycosyl moiety directly (i.e., through a single group formed by the reaction of two reactive groups) or through a linker moiety, e.g., substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, etc. The polymer can be attached at any position of a glycosyl moiety of Factor VIII. The glycosyl moiety can be N-linked or O-linked glycosylation. For example, the glycosyl moiety can be O-linked glycosylation, and the polymer moiety can be attached to the O-linked glycosyl moiety. See US2008/0280818 and WO 2009/089396, each of which is incorporated herein by reference in its entirety.

In other embodiments, Factor VIII used in the invention is conjugated to one or more polymers. The polymer can be water-soluble and covalently or non-covalently attached to Factor VIII or other moieties conjugated to Factor VIII. Non-limiting examples of the polymer can be poly(alkylene oxide), poly(vinyl pyrrolidone), poly(vinyl alcohol), polyoxazoline, or poly(acryloylmorpholine). Additional types of polymer-conjugated Factor VIII are disclosed in U.S. Pat. No. 7,199,223, which is disclosed by reference in its entirety.

In certain embodiments, Factor VIII is fused to albumin or albumin fragment or variant. Non-limiting examples of albumin-fused Factor VIII is disclosed in U.S. Application publication no. 2008/0261877, which is incorporated herein by reference in its entirety. The Factor VIII polypeptide can be fused to either the N-terminal end of an albumin or to the C-terminal end of the albumin, provided the Factor VIII component of the Factor VIII-albumin fusion protein can be processed by an enzymatically-active proprotein convertase to yield a processed Factor VIII-containing polypeptide, as described herein. In one embodiment the Factor VIII polypeptide is a human Factor VIII polypeptide and the albumin polypeptide is a human albumin polypeptide.

In some embodiments, the Factor VIII protein is fused to a transferrin protein. A Factor VIII-transferrin fusion protein as used herein refers to a fusion protein that includes a Factor VIII (full-length or B-domain-deleted) polypeptide covalently bonded to transferrin. The Factor VIII polypeptide can be fused to either the N-terminal end of an transferrin polypeptide or to the C-terminal end of an transferrin polypeptide, provided the Factor VIII component of the Factor VIII-transferrin fusion protein can be processed by an enzymatically-active proprotein convertase to yield a processed Factor VIII-containing polypeptide, as described herein. In one embodiment the Factor VIII polypeptide is a human Factor VIII polypeptide and the transferrin polypeptide is a human transferrin polypeptide.

In some embodiments, the Factor VIII protein is fused to an artificial protein, such as an XTEN sequence (Schellenberger et al. Nature Biotechnology 2009. 27:1186).

The Factor VIII and a heterologous polypeptide, e.g., an immunoglobulin constant region (e.g., an Fc domain), albumin, or transferrin, may be fused by a direct peptide bond or by a linker. The linker can comprise any organic molecule. In one embodiment, the linker is polyethylene glycol (PEG). In another embodiment, the linker is amino acids. The linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids. In one embodiment, the linker is the eight amino acid linker, EFAGAAAV (SEQ ID NO: 50). Any of the linkers described herein may be used in the Factor VIII fusion, e.g., a monomer-dimer hybrid, including EFAGAAAV (SEQ ID NO: 50).

The linker can comprise the sequence G_(n). The linker can comprise the sequence (GA)_(n). The linker can comprise the sequence (GGS)_(n). The linker can comprise the sequence (GGS)_(n)(GGGGS)_(n) (SEQ ID NO: 63). In these instances, n may be an integer from 1-10, i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Examples of linkers include, but are not limited to, GGG, SGGSGGS (SEQ ID NO: 51), GGSGGSGGSGGSGGG (SEQ ID NO: 52), GGSGGSGGGGSGGGGS (SEQ ID NO: 53), GGSGGSGGSGGSGGSGGS (SEQ ID NO: 54). The linker does not eliminate or diminish the clotting activity of Factor VIII fusion protein. Optionally, the linker enhances the clotting activity of Factor VIII fusion protein, e.g., by further diminishing the effects of steric hindrance and making the Factor VIII portion more accessible to its target binding site.

In one embodiment, the linker for Factor VIII fusion is 15-25 amino acids long. In another embodiment, the linker for Factor VIII fusion is 15-20 amino acids long. In some embodiments, the linker for Factor VIII fusion is 10-25 amino acids long. In other embodiments, the linker for Factor VIII fusion is 15 amino acids long. In still other embodiments, the linker for Factor VIII fusion is (GGGGS)_(n) (SEQ ID NO: 55) where G represents glycine, S represents serine and n is an integer from 1-10. In a specific embodiment, n is 3 (SEQ ID NO: 56).

The linker may also incorporate a moiety capable of being cleaved either chemically (e.g., hydrolysis of an ester bond), enzymatically (i.e., incorporation of a protease cleavage sequence) or photolytically (e.g., a chromophore such as 3-amino-3-(2-nitrophenyl) proprionic acid (ANP)) in order to release the biologically active molecule from the Fc protein.

Proprotein Convertases (Also Known as Prohormone Convertase)

The invention is based on the discovery that a proprotein convertase is capable of processing Factor VIII or Factor VIII variants at the corresponding residue by activating nonprocessed Factor VIII into processed Factor VIII. Proprotein convertases are also known as proprotein convertase subtilisin/kexin type proteins (PCSK), and there are nine subtypes of proprotein convertases (PCSK1, PCSK2, PCSK3, PCSK4, PCSK5, PCSK6, PCSK7, PCSK8, and PCSK9). In one embodiment, the proprotein convertase used for the present invention is selected from the group consisting of PCSK1, PCSK2, PCSK3, PCSK4, PCSK5, PCSK6, PCSK7, PCSK8, PCSK9, and a yeast Kex 2 protein. The proprotein convertase used for the purpose of this invention is an exogenous enzyme that is either added as a separate protein or expressed in a separate nucleotide sequence in the host cell expressing Factor VIII or expressed separately in a host cell different from the host cell expressing Factor VIII.

Proprotein convertase subtilisin/kexin type 1 (PCSK1) is also known as proprotein convertase 1(PC1), proprotein convertase 3 (PC3), PC1/3, neuroendocrine convertase 1 (NEC 1), body mass index quantitative trait locus (BMIQ12), or prohormone convertase 1. The gene encoding PCSK1 is known as Pcsk1, Att-1, Nec-1, or Nec1 and has Accession Number M90753.1 in GenBank. The full-length human PCSK1 polypeptide has Accession Number P29120 in UniProtKB/Swiss-Prot entry and consists of a signal peptide (amino acids 1-27), a propeptide (amino acids 28-110), and the active protein domain (amino acids 111-753) including the catalytic domain (amino acids 122-410). The nucleotide and amino acid sequences of PCSK1 are represented herein as SEQ ID NO: 7 and SEQ ID NO: 8, respectively. Variants of human PCSK1 include, but are not limited to, the polypeptides with the following mutations: R80Q, N221D, S307L, G483R, Q665E, S690T, S357G, or S690T. Also included is PCSK1 from a different species, e.g., mouse, rat, monkey, dog, drosophila, or porcine.

PCSK1 Amino Acid Sequence (SEQ ID NO: 8) MERRAWSLQC TAFVLFCAWC ALNSAKAKRQ FVNEWAAEIP GGPEAASAIA EELGYDLLGQ IGSLENHYLF KHKNHPRRSR RSAFHITKRL SDDDRVIWAE QQYEKERSKR SALRDSALNL FNDPMWNQQW YLQDTRMTAA LPKLDLHVIP VWQKGITGKG VVITVLDDGL EWNHTDIYAN YDPEASYDFN DNDHDPFPRY DPTNENKHGT RCAGEIAMQA NNHKCGVGVA YNSKVGGIRM LDGIVTDAIE ASSIGFNPGH VDIYSASWGP NDDGKTVEGP GRLAQKAFEY GVKQGRQGKG SIFVWASGNG GRQGDNCDCD GYTDSIYTIS ISSASQQGLS PWYAEKCSST LATSYSSGDY TDQRITSADL HNDCTETHTG TSASAPLAAG IFALALEANP NLTWRDMQHL VVWTSEYDPL ANNPGWKKNG AGLMVNSRFG FGLLNAKALV DLADPRTWRS VPEKKECVVK DNDFEPRALK ANCEVIIETP TRACEGQENA IKSLEHVQFE ATIEYSRRGD LEVTLTSAAG TSTVLLAERE RDTSPNGFKN WDFMSVHTWG ENPIGTWTLR ITDMSGRIQN EGRIVNWKLI LHGTSSQPEH MKQPRVYTSY NTVQNDRRGV EKMVDPGEEQ PTQENPKENT LVSKSPSSSS VGGRRDELEE GAPSQAMLRL LQSAFSENSP PKQSPKKSPS AKLNIPYENF YEALEKLNKP SQLKDSEDSL YNDYVDVFYN TKPYKHRDDR LLQALVDILN EEN PCSK 1 Nucleic Acid Sequence (SEQ ID NO: 7) atggagcgaa gagcctggag tctgcagtgc actgctttcg tcctcttttg cgcttggtgt gcactgaaca gtgcaaaagc gaaaaggcaa tttgtcaatg aatgggcagc ggagatcccc gggggcccgg aagcagcctc ggccatcgcc gaggagctgg gctatgacct tttgggtcag attggttcac ttgaaaatca ctacttattc aaacataaaa accaccccag aaggtctcga aggagtgcct ttcatatcac taagagatta tctgatgatg atcgtgtgat atgggctgaa caacagtatg aaaaagaaag aagtaaacgt tcagctctaa gggactcagc actaaatctc ttcaatgatc ccatgtggaa tcagcaatgg tacttgcaag ataccaggat gacggcagcc ctgcccaagc tggaccttca tgtgatacct gtttggcaaa aaggcattac gggcaaagga gttgttatca ccgtactgga tgatggtttg gagtggaatc acacggacat ttatgccaac tatgatccag aggctagcta tgattttaat gataatgacc atgatccatt tccccgatat gatcccacaa acgagaacaa acacgggacc agatgtgcag gagaaattgc catgcaagca aataatcaca aatgcggggt tggagttgca tacaattcca aagttggagg cataagaatg ctggatggca ttgtgacgga tgctattgag gccagttcaa ttggattcaa tcctggacac gtggatattt acagtgcaag ctggggccct aatgatgatg ggaaaactgt ggaggggcct ggccggctag cccagaaggc ttttgaatat ggtgtcaaac aggggagaca ggggaagggg tccatcttcg tctgggcttc gggaaacggg gggcgtcagg gagataattg tgactgtgat ggctacacag acagcatcta caccatctcc atcagcagtg cctcccagca aggcctatcc ccctggtacg ctgagaagtg ctcctccaca ctggccacct cttacagcag cggagattac accgaccaga gaatcacgag cgctgacctg cacaatgact gcacggagac gcacacaggc acctcggcct ctgcacctct ggctgctggc atcttcgctc tggccctgga agcaaaccca aatctcacct ggcgagatat gcagcacctg gttgtctgga cctctgagta tgacccgctg gccaataacc ctggatggaa aaagaatgga gcaggcttga tggtgaatag tcgatttgga tttggcttgc taaatgccaa agctctggtg gatttagctg accccaggac ctggaggagc gtgcctgaga agaaagagtg tgttgtaaag gacaatgact ttgagcccag agccctgaaa gctaatggag aagttatcat tgaaattcca acaagagctt gtgaaggaca agaaaatgct atcaagtccc tggagcatgt acaatttgaa gcaacaattg aatattcccg aagaggagac cttcatgtca cacttacttc tgctgctgga actagcactg tgctcttggc tgaaagagaa cgggatacat ctcctaatgg ctttaagaac tgggacttca tgtctgttca cacatgggga gagaacccta taggtacttg gactttgaga attacagaca tgtctggaag aattcaaaat gaaggaagaa ttgtgaactg gaagctgatt ttgcacggga cctcttctca gccagagcat atgaagcagc ctcgtgtgta cacgtcctac aacactgttc agaatgacag aagaggggtg gagaagatgg tggatccagg ggaggagcag cccacacaag agaaccctaa ggagaacacc ctggtgtcca aaagccccag caggaggagc gtagggggcc ggagggatga gttggaggag ggagcccctt cccaggccat gctgcgactc ctgcaaagtg ctttcagtaa aaactcaccg ccaaagcaat caccaaagaa gtccccaagt gcaaagctca acatccctta tgaaaacttc tacgaagccc tggaaaagct gaacaaacct tcccagctta aagactctga agacagtctg tataatgact atgttgatgt tttttataac actaaacctt acaagcacag agacgaccgg ctgcttcaag ctctggtgga cattctgaat gaggaaaatt aa

In one embodiment, the proprotein convertase used for the present invention comprises an amino acid sequence, which is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 122-410 of SEQ ID NO: 8, amino acids 111 to 753 of SEQ ID NO: 8, amino acids 28 to 753 of SEQ ID NO: 8, or SEQ ID NO: 8, wherein the amino acid sequence is capable of cleaving full-length Factor VIII at amino acid residue 1648.

Proprotein convertase subtilisin/kexin type 2 (PCSK2) is also known as proprotein convertase 2(PC2), neuroendocrine convertase 2 (NEC2), KEX2-like endoprotease 2, or prohormone convertase 2. The gene encoding PCSK2 is known as pcsk2 or nec2 has Accession Number AH002912 in GenBank. The full-length human PCSK2 polypeptide has Accession Number P16519 in UniProtKB/Swiss-Prot entry and consists of a signal peptide (amino acids 1-25), a propeptide (amino acids 26-109), and the active protein domain (amino acids 110-638) including the catalytic domain (amino acids 122-415). The nucleotide and amino acid sequences of PCSK2 are represented herein as SEQ ID NO: 9 and SEQ ID NO: 10, respectively. Variants of human PCSK2 include, but are not limited to, the polypeptides with the following mutations: D424N or EL283-284DV. Also included is PCSK2 from a different species, e.g., mouse, rat, monkey, dog, drosophila, or porcine.

PCSK2 Amino Acid Sequence (SEQ ID NO: 10) MKGGCVSQWK AAAGFLFCVM VFASAERPVF TNHFLVELHK GGEDKARQVA AEHGFGVRKL PFAEGLYHFY HNGLAKAKRR RSLHHKQQLE RDPRVKMALQ QEGFDRKKRG YRDINEIDIN MNDPLFTKQW YLINTGQADG TPGLDLNVAE AWELGYTGKG VTIGIMDDGI DYLEPDLASN YNAEASYDFS SNDPYPYPRY TDDWFNSHGT RCAGEVSAAA NNNICGVGVA YNSKVAGIRM LDQPFMTDII EASSISHMPQ LIDIYSASWG PTDNGKTVDG PRELTLQAMA DGVNKGRGGK GSTYVWASGD GGSYDDCNCD GYASSMWTIS INSAINDGRT ALYDESCSST LASTFSNGRK RNPEAGVATT DLYGNCTLRH SGTSAAAPEA AGVFALALEA NLGLTWRDMQ HLTVLTSKRN QLHDEVHQWR RNGVGLEFNH LEGYGVLDAG AMVKMAKDWK TVPERFHCVG GSVQDPEKIP STGKLVLTLT TDACEGKENF VRYLEHVQAV ITVNATRRGD LNINMTSPMG TKSILLSRRP RDDDSKVGFD KWPFMTTHTW GEDARGTWTL ELGFVGSAPQ KGVLKEWTLM LEGTQSAPYI DQVVRDYQSK LAMSKKEELE EELDEAVERS LKSTLNKN PCSK 2 Nucleic Acid Sequence (SEQ ID NO: 9) atgaagggtg gttgtgtctc ccagtggaag gcggccgccg ggttcctctt ctgtgtcatg gtttttgcat ctgctgagcg accggtcttc acgaatcatt ttcttgtgga gttgcataaa gggggagagg acaaagctcg ccaagttgca ggagaacacg gctttggagt ccgaaagctt ccctttgctg aaggtctgta ccacttttat cacaatggcc ttgcaaaggc caagagaaga cgcagcctac accacaagca gcagctggag agagacccca gggtaaagat ggctttgcag caggaaggat ttgaccgaaa aaagcgaggt tacagagaca tcaatgagat cgacatcaac atgaacgatc ctctttttac aaagcagtgg tatctgatca atactgggca agctgatggc actcctggcc ttgatttgaa tgtggctgaa gcctgggagc tgggatacac agggaaaggt gttaccattg gaattatgga tgatgggatt gactatctcc acccggacct ggcctccaac tataatgccg aagcaagtta cgacttcagc agcaacgacc cctatcctta ccctcggtac acagatgact ggtttaacag ccacgggacc cgatgtgcag gagaagtttc tgctgccgcc aacaacaata tctgtggagt tggagtagca tacaactcca aggttgcagg catccggatg ctggaccagc cattcatgac agacatcatc gaggcctcct ccatcagtca tatgccacag ctgattgaca tctacagcgc cagctggggc cccacagaca acggcaagac agtggatggg ccccgggagc tcacgctgca ggccatggcc gatggcgtga acaagggccg cggcggcaaa ggcagcatct acgtgtgggc ctccggggac ggcggcagct atgacgactg caactgcgac ggctacgcct ccagcatgtg gaccatctcc atcaactcag ccatcaacga cggcaggact gccctgtacg acgagagctg ctcttccacc ttggcttcca ccttcagcaa cgggaggaaa aggaacccgg aggccggtgt ggcaaccaca gatttgtacg gcaactgcac tctgaggcat tctgggacat ctgcagctgc ccccgaggca gctggtgtgt ttgcactggc tctggaggct aacctgggtc tgacctggcg ggacatgcag catctgactg tgctcacctc caaacggaac cagcttcacg acgaggtcca tcagtggcgg cgcaatgggg tcggcctgga atttaatcac ctctttggct acggggtcct tgatgcaggt gccatggtga aaatggctaa agactggaaa accgtgcctg agagattcca ctgtgtggga ggctccgtgc aggaccctga gaaaatacca tccactggca agttggtgct gacactcaca accgacgcct gtgaggggaa ggaaaatttt gtccgctacc tggagcatgt ccaggctgtc atcacggtca acgcaaccag aagaggagac ctgaacatca acatgacttc ccctatgggc accaagtcca ttttgctgag ccggcgtcca agggatgacg actccaaggt gggctttgac aagtggcctt tcatgaccac tcacacgtgg ggggaagagg cccgaggcac ctggaccctg gagctgggat ttgtcggcag cgccccgcag aagggggtgc tgaaggagtg gaccctgatg ctgcatggca ctcagagtgc ccggtacatc gaccaggtgg tgcgggatta ccagtccaag ttggccatgt ccaagaaaga ggagctggag gaagagctgg acgaagccgt ggagagaagc ctgaaaagca tccttaacaa gaactag

In one embodiment, the proprotein convertase used for the present invention comprises an amino acid sequence, which is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 122-415 of SEQ ID NO: 10, amino acids 110 to 638 of SEQ ID NO: 10, amino acids 26 to 638 of SEQ ID NO: 10, or SEQ ID NO: 10, wherein the amino acid sequence is capable of cleaving full-length Factor VIII at amino acid residue 1648.

Proprotein convertase subtilisin/kexin type 3 (PCSK3) is also known as Furin, Paired Basic Amino Acid Residue Cleaving Enzyme (PACE), Dibasic-Processing Enzyme, or Prohormone Convertase 3. The gene encoding PCSK3 is known as Pcsk3, Furin, or Fur and has Accession Number BC012181 in GenBank. The full-length human PCSK3 polypeptide has Accession Number P09958 in UniProtKB/Swiss-Prot entry and consists of a signal peptide (amino acids 1-24), propeptide (amino acids 25-107), and the active protein domain (amino acids 108-794). The nucleotide and amino acid sequences of PCSK3 are represented herein as SEQ ID NO: 11 and SEQ ID NO: 12, respectively. Variants of human PCSK3 include, but are not limited to, the polypeptides with the following mutations: A43V or W547R. Also included is PCSK3 from a different species, e.g., mouse, rat, monkey, dog, drosophila, or porcine.

PCSK3 Amino Acid Sequence (SEQ ID NO: 12) MELRPWLLWV VAATGTLVLL AADAQGQKVF TNTWAVRIPG GPAVANSVAR KHGELNLGQI FGDYYHFWHR GVTKRSLSPH RPRHSRLQRE PQVQWLEQQV AKRRTKRDVY QEPTDPKFPQ QWYLSGVTQR DLNVKAAWAQ GYTGHGIVVS ILDDGIEKNH PDLAGNYDPG ASFDVNDQDP DPQPRYTQMN DNRHGTRCAG EVAAVANNGV CGVGVAYNAR IGGVRMLDGE VTDAVEARSL GLNPNHIHIY SASWGPEDDG KTVDGPARLA EEAFFRGVSQ GRGGLGSIFV WASGNGGREH DSCNCDGYTN SIYTLSISSA TQFGNVPWYS EACSSTLATT YSSGNQNEKQ IVTTDLRQKC TESHTGTSAS APLAAGIIAL TLEANKNLTW RDMQHLVVQT SKPAHLNAND WATNGVGRKV SHSYGYGLLD AGAMVALAQN WTTVAPQRKC IIDILTEPKD IGKRLEVRKT VTACLGEPNH ITRLEHAQAR LTLSYNRRGD LAIHLVSPMG TRSTLLAARP HDYSADGFND WAFMTTHSWD EDPSGEWVLE IENTSEANNY GTLTKFTLVL YGTAPEGLPV PPESSGCKTL TSSQACVVCE EGFSLHQKSC VQHCPPGFAP QVLDTHYSTE NDVETIRASV CAPCHASCAT CQGPALTDCL SCPSHASLDP VEQTCSRQSQ SSRESPPQQQ PPRLPPEVEA GQRLRAGLLP SHLPEVVAGL SCAFIVLVFV TVFLVLQLRS GESERGVKVY TMDRGLISYK GLPPEAWQEE CPSDSEEDEG RGERTAFIKD QSAL PCSK 3 Nucleic Acid Sequence (SEQ ID NO: 11) atggagctga ggccctggtt gctatgggtg gtaggagcaa caggaacctt ggtcctgcta gcagctgatg ctcagggcca gaaggtcttc accaacacgt gggctgtgcg catccctgga ggcccagcgg tggccaacag tgtggcacgg aagcatgggt tcctcaacct gggccagatc ttcggggact attaccactt ctggcatcga ggagtgacga agcggtccct gtcgcctcac cgcccgcggc acagccggct gcagagggag cctcaagtac agtggctgga acagcaggtg gcaaagcgac ggactaaacg ggacgtgtac caggagccca cagaccccaa gtttcctcag cagtggtacc tgtctggtgt cactcagcgg gacctgaatg tgaaggcggc ctgggcgcag ggctacacag ggcacggcat tgtggtctcc attctggacg atggcatcga gaagaaccac ccggacttgg caggcaatta tgatcctggg gccagttttg atgtcaatga ccaggaccct gacccccagc ctcggtacac acagatgaat gacaacaggc acggcacacg gtgtgcgggg gaagtggctg cggtggccaa caacggtgtc tgtggtgtag gtgtggccta caacgcccgc attggagggg tgcgcatgct ggatggcgag gtgacagatg cagtggaggc acgctcgctg ggcctgaacc ccaaccacat ccacatctac agtgccagct ggggccccga ggatgacggc aagacagtgg atgggccagc ccgcctcgcc gaggaggcct tcttccgtgg ggttagccag ggccgagggg ggctgggctc catctttgtc tgggcctcgg ggaacggggg ccgggaacat gacagctgca actgcgacgg ctacaccaac agtatctaca cgctgtccat cagcagcgcc acgcagtttg gcaacgtgcc gtggtacagc gaggcctgct cgtccacact ggccacgacc tacagcagtg gcaaccagaa tgagaagcag atcgtgacga ctgacttgcg gcagaagtgc acggagtctc acacgggcac ctcagcctct gcccccttag cagccggcat cattgctctc accctggagg ccaataagaa cctcacatgg cgggacatgc aacacctggt ggtacagacc tcgaagccag cccacctcaa tgccaacgac tgggccacca atggtgtggg ccggaaagtg agccactcat atggctacgg gcttttggac gcaggcgcca tggtggccct ggcccagaat tggaccacag tggcccccca gcggaagtgc atcatcgaca tcctcaccga gcccaaagac atcgggaaac ggctcgaggt gcggaagacc gtgaccgcgt gcctgggcga gcccaaccac atcactcggc tggagcacgc tcaggcgcgg ctcaccctgt cctataatcg ccgtggcgac ctggccatcc acctggtcag ccccatgggc acccgctcca ccctgctggc agccaggcca catgactact ccgcagatgg gtttaatgac tgggccttca tgacaactca ttcctgggat gaggatccct ctggcgagtg ggtcctagag attgaaaaca ccagcgaagc caacaactat gggacgctga ccaagttcac cctcgtactc tatggcaccg cccctgaggg gctgcccgta cctccagaaa gcagtggctg caagaccctc acgtccagtc aggcctgtgt ggtgtgcgag gaaggcttct ccctgcacca gaagagctgt gtccagcact gccctccagg cttcgccccc caagtcctcg atacgcacta tagcaccgag aatgacgtgg agaccatccg ggccagcgtc tgcgccccct gccacgcctc atgtgccaca tgccaggggc cggccctgac agactgcctc agctgcccca gccacgcctc cttggaccct gtggagcaga cttgctcccg gcaaagccag agcagccgag agtccccgcc acagcagcag ccacctcggc tgcccccgga ggtggaggcg gggcaacggc tgcgggcagg gctgctgccc tcacacctgc ctgaggtggt ggccggcctc agctgcgcct tcatcgtgct ggtcttcgtc actgtcttcc tggtcctgca gctgcgctct ggctttagtt ttcggggggt gaaggtgtac accatggacc gtggcctcat ctcctacaag gggctgcccc ctgaagcctg gcaggaggag tgcccgtctg actcagaaga ggacgagggc cggggcgaga ggaccgcctt tatcaaagac cagagcgccc tctga

In one embodiment, the proprotein convertase used for the present invention comprises an amino acid sequence, which is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 108 to 794 of SEQ ID NO: 12, amino acids 25 to 794 of SEQ ID NO: 12, or SEQ ID NO: 12, wherein the amino acid sequence is capable of cleaving full-length Factor VIII at amino acid residue 1648.

Proprotein convertase subtilisin/kexin type 4 (PCSK4) is also known as proprotein convertase 4 (PC4), Neuroendocrine convertase 3 (NEC3), prohormone convertase 3, or KEX2-like endoprotease 3. The gene encoding PCSK4 is known as Pcsk3, Nec-3, or Nec3 and has Accession Number AY358963 in GenBank. The full-length human PCSK3 polypeptide has Accession Number AAQ89322.1 in GenBank and consists of a signal peptide (amino acids 1-25), propeptide (amino acids 26-113), and the active protein domain (amino acids 114-755) including the catalytic domain (amino acids 124-417). The nucleotide and amino acid sequences of PCSK4 are represented herein as SEQ ID NO: 13 or 15 and SEQ ID NO: 14 or 16, respectively. Variants of human PCSK4 include, but are not limited to, the polypeptides with the mutation of T267M. Also included is PCSK4 from a different species, e.g., mouse, rat, monkey, dog, drosophila, or porcine.

PCSK4 Isoform 1 Amino Acid Sequence (SEQ ID NO: 14) MRPAPIALWL RLVLALALVR PRAVGWAPVR APIYVSSWAV QVSQGNREVE RLARKFGFVN LGPIFSDGQY FHLRHRGVVQ QSLTPHWGHR LHLKKNPKVQ WFQQQTLQRR VKRSVVVPTD PWFSKQWYMN SEAQPDLSIL QAWSQGLSGQ GIVVSVLDDG TEKDHPDLWA NYDPLASYDF NDYDPDPQPR YTPSKENRHG TRCAGEVAAM ANNGFCGVGV AFNARIGGVR MLDGTITDVI EAQSLSLQPQ HIHIYSASWG PEDDGRTVDG PGIITREAFR RGVTKGRGGL GTLFIWASGN GGLHYDNCNC DGYTNSIHTL SVGSTTQQGR VPWYSEACAS TLTTTYSSGV ATDPQIVTTD LHHGCTDQHT GTSASAPLAA GMIALALEAN PFLTWRDMQH LVVRASKPAH LQAEDWRTNG VGRQVSEHYG YGLLDAGLLV DTARTWLPTQ PQRKCAVRVQ SRPTPILPLI YIRENVSACA GLHNSIRSLE HVQAQLTLSY SPRGDLEISL TSPMCTRSTL VAIRPLDVST EGYNNWVFMS THFWDENPQG VWTLGLENKG YYFNTGTLYR YTLLLYGTAE DMTARPTGPQ VTSSACVQRD TEGLCQACDG PAYILGQLCL AYCPPRFFNH TRLVTAGPGH TAAPALRVCS SCHASCYTCR GGSPRDCTSC PPSSTLDQQQ GSCMGPTTPD SRPRLRAAAC PHHRCPASAM VLSLLAVTLG GPVLCGMSMD LPLYAWLSRA RATPTKPQVW LPAGT PCSK 4 Isoform 1 Nucleic Acid Sequence (SEQ ID NO: 13) atgcggcccg ccccgattgc gctgtggctg cgcctggtct tggccctggc ccttgtccgc ccccgggctg tggggtgggc cccggtccga gcccccatct atgtcagcag ctgggccgtc caggtgtccc agggtaaccg ggaggtcgag cgcctggcac gcaaattcgg cttcgtcaac ctggggccga tcttctctga cgggcagtac tttcacctgc ggcaccgggg cgtggtccag cagtccctga ccccgcactg gggccaccgc ctgcacctga agaaaaaccc caaggtgcag tggttccagc agcagacgct gcagcggcgg gtgaaacgct ctgtcgtggt gcccacggac ccctggttct ccaagcagtg gtacatgaac agcgaggccc aaccagacct gagcatcctg caggcctgga gtcaggggct gtcaggccag ggcatcgtgg tctctgtgct ggacgatggc atcgagaagg accacccgga cctctgggcc aactacgacc ccctggccag ctatgacttc aatgactacg acccggaccc ccagccccgc tacaccccca gcaaagagaa ccggcacggg acccgctgtg ctggggaggt ggccgcgatg gccaacaatg gcttctgtgg tgtgggggtc gctttcaacg cccgaatcgg aggcgtacgg atgctggacg gtaccatcac cgatgtcatc gaggcccagt cgctgagcct gcagccgcag cacatccaca tttacagcgc cagctggggt cccgaggacg acggccgcac ggtggacggc cccggcatcc tcacccgcga ggccttccqg cgtggtgtga ccaagggccg cggcgggctg ggcacgctct tcatctgggc ctcgggcaac ggcqgcctgc actacgacaa ctgcaactgc gacggctaca ccaacagcat ccacacgctt tccgtgggca gcaccaccca gcagggccgc gtgccctggt acagcgaagc ctgcgcctcc accctcacca ccacctacag cagcggcgtg gccaccgacc cccagatcgt caccacggac ctgcatcacg ggtgcacaga ccagcacacg ggcacctcgg cctcagcccc actggcggcc ggcatgatcg ccctagcgct ggaggccaac ccgttcctga cgtggagaga catgcagcac ctggtggtcc gcgcgtccaa gccggcgcac ctgcaggccg aggactggag gaccaacggc gtggggcgcc aagtgagcca tcactacgga tacgggctgc tggacgccgg gctgctggtg gacaccgccc gcacctggct gcccacccag ccgcagagga agtgcgccgt ccgggtccag agccgcccca cccccatcct gccgctgatc tacatcaggg aaaacgtatc ggcctgcgcc ggcctccaca actccatccg ctcgctggag cacgtgcagg cgcagctgac gctgtcctac agccggcgcg gagacctgga gatctcgctc accagcccca tgggcacgcg ctccacactc gtggccatac gacccttgga cgtcagcact gaaggctaca acaactgggt cttcatgtcc acccacttct gggatgagaa cccacagggc gtgtggaccc tgggcctaga gaacaagggc tactatttca acacggggac gttgtaccgc tacacgctgc tgctctatgg gacggccgag gacatgacag cgcggcctac aggcccccag gtgaccagca gcgcgtgtgt gcagcgggac acagaggggc tgtgccaggc gtgtgacggc cccgcctaca tcctgggaca gctctgcctg gcctactgcc ccccgcggtt cttcaaccac acaaggctgg tgaccgctgg gcctgggcac acggcggcgc ccgcgctgag ggtctgctcc agctgccatg cctcctgcta cacctgccgc ggcggctccc cgagggactg cacctcctgt cccccatcct ccacgctgga ccagcagcag ggctcctgca tgggacccac cacccccgac agccgccccc ggcttagagc tgccgcctgt ccccaccacc gctgcccagc ctcggccatg gtgctgagcc tcctggccgt gaccctcgga ggccccgtcc tctgcggcat gtccatggac ctcccactat acgcctggct ctcccgtgcc agggccaccc ccaccaaacc ccaggtctgg ctgccagctg gaacctga PCSK 4 Isoform 2 Amino Acid Sequence (SEQ ID NO: 16) MGTRSTLVAI RPLDVSTEGY NNWVEMSTHF WDENPQGVWT LGLENKCYYF NTGTLYRYTL LLYGTAEDMT ARPTGPQVTS SACVQRDTEG LCQACDGPAY ILGQLCLAYC PPRFFNHTRL VTAGPCHTAA PALRVCSSCH ASCYTCRGGS PRDCTSCPPS STLDQQQGSC MGPTTPDSRP RLRAAACPHH RCPASAMVLS LLAVTLGGPV LCGMSMDLPL YAWLSRARAT PTKPQVWLPA GT PCSK 4 isoform 2 Nucleic Acid Sequence (SEQ ID NO: 15) atgggcacgc gctccacact cgtggccata cgacccttgg acgtcagcac tgaaggctac aacaactggg tcttcatgtc cacccacttc tgggatgaga acccacaggg cgtgtggacc ctgggcctag agaacaaggg ctactatttc aacacgggga cgttgtaccg ctacacgctg ctgctctatg ggacggccga ggacatgaca gcgcggccta caggccccca ggtgaccagc agcgcgtgtg tgcagcggga cacagagggg ctgtgccagg cgtgtgacgg ccccgcctac atcctgggac agctctgcct ggcctactgc cccccgcggt tcttcaacca cacaaggctg gtgaccgctg ggcctgggca cacggcggcg cccgcgctga gggtctgctc cagctgccat gcctcctgct acacctgccg cggcggctcc ccgagggact gcacctcctg tcccccatcc tccacgctgg accagcagca gggctcctgc atgggaccca ccacccccga cagccgcccc cggcttagag ctgccgcctg tccccaccac cgctgcccag cctcggccat ggtgctgagc ctcctggccg tgaccctcgg aggccccgtc ctctgcggca tgtccatgga cctcccacta tacgcctggc tctcccgtgc cagggccacc cccaccaaac cccaggtctg gctgccagct ggaacctga

In one embodiment, a proprotein convertase used for the present invention comprises an amino acid sequence, which is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 124 to 417 of SEQ ID NO: 14, amino acids 114-755 of SEQ ID NO: 14, amino acids 26 to 755 of SEQ ID NO: 14, SEQ ID NO: 14, amino acids 114-513 of SEQ ID NO: 14, amino acids 26 to 513 of SEQ ID NO: 14 or SEQ ID NO: 16, wherein the amino acid sequence is capable of cleaving full-length Factor VIII at amino acid residue 1648.

Proprotein convertase subtilisin/kexin type 5 (PCSK5) is also known as proprotein convertase 5 (PC5), proprotein convertase 6 (PC6), subtilisin/kexin-like protease PC5, or subtilisin-like proprotein convertase 6 (SPC6). The gene encoding PCSK5 is known as Pcsk5, furl, or PC5 and has Accession Number AL834522.1 in GenBank. The full-length human PCSK5 polypeptide has Accession Number NM-006200 in GenBank and consists of a signal peptide (amino acids 1-32), propeptide (amino acids 33-114), and the active protein domain (amino acids 115-913) including a catalytic domain (amino acids 115-454) and a cysteine-rich motif (amino acids 636-868). The nucleotide and amino acid sequences of PCSK5 are represented herein as SEQ ID NO: 17 and SEQ ID NO: 18, respectively. Variants of human PCSK5 include, but are not limited to, the polypeptides with the following mutations: G2D, G4E, F118S, A121V, R511A, and Q601R. An isoform is also available in GenBank as Accession No. NP_001177411 (SEQ ID NO: 57) for the amino acid sequence and Accession No. NM_001190482 (SEQ ID NO: 58) for the nucleotide sequence, both of which are incorporated herein by reference in its entirety. Also included is PCSK5 from a different species, e.g., mouse, rat, monkey, dog, drosophila, or porcine.

PCSK5 Amino Acid Sequence (SEQ ID NO: 18) MGWGSRCCCP GRLDLLCVLA LLGGCLLPVC RTRVYTNHWA VKIAGGFPEA NRTASKYGFI NIGQIGALKD YYHFYHSRTI KRSVISSRGT HSFISMEPKV EWIQQQVVKK RTKRDYDFSR AQSTYFNDPK WPSMWYMHCS DNTHPCQSDM NIEGAWKRGY TGKNIVVTIL DDGIERTHPD LMQNYDALAS CDVNGNDLDP MPRYDASNEN KHGTRCAGEV AAAANNSHCT VGIAFNAKIG GVRMLDGDVT DMVEAKSVSF NPQHVHIYSA SWCPDDDGKT VDGPAPLTRQ AFENGVRMGR RGLGSVFVWA SGNCCRSKDH CSCDGYTNSI YTISISSTAE SGKKPWYLEE CSSTLATTYS SGESYDKKII TTDLRQRCTD NHTGTSASAP MAAGIIALAL EANPFLTWRD VQHVIVRTSR AGHLNANDWK TNAAGFKVSH LYGFGLMDAE AMVMEAEKWT TVPRQHVCVE STDRQIKTIR PNSAVRSIYK ASGCSDNPNR HVNYLEHVVV RITITHPRRG DLAIYLTSPS GTRSQLLANR LFDESMEGFK NWEFMTIHCW GERAAGDWVL EVYDTPSQLR NFKTPGKLKE WSLVLYGTSV QPYSPTNEFP KVERFRYSRV EDPTDDYGTE DYAGPCDPEC SEVGCDGPGP DHCNDCLHYY YKLKNNTRIC VSSCPPGHYE ADKKRCRKCA PNCESCFGSH GDQCMSCKYG YFLNEETNSC VTHCPDGSYQ DTKKNLCRKC SENCKTCTEF HNCTECRDGL SLQGSRCSVS CEDGRYFNGQ DCQPCHRFCA TCAGAGADGC INCTEGYFME DGRCVQSCSI SYYFDHSSEN GYKSCKKCDI SCLTCNGPGE KNCTSCPSGY LLDLGMCQMG AICKDATEES WAEGGFCMLV KKNNLCQRKV LQQLCCKTCT FQG PCSK 5 Nucleic Acid Sequence (SEQ ID NO: 17) atgggctggg ggagccgctg ctgctgcccg ggacgtttgg acctgctgtg cgtgctggcg ctgctcgggg gctgcctgct ccccgtgtgt cggacgcgcg tctacaccaa ccactgggca gtcaaaatcg ccgggggctt cccggaggcc aaccgtatcg ccagcaagta cggattcatc aacataggac agataggggc cctgaaggac tactaccact tctaccatag caggacgatt aaaaggtcag ttatctcgag cagagggacc cacagtttca tttcaatgga accaaaggtg gaatggatcc aacagcaagt ggtaaaaaag cggacaaaga gggattatga cttcagtcgt gcccagtcta cctatttcaa tgatcccaag tggcccagca tgtggtatat gcactgcagt gacaatacac atccctgcca gtctgacatg aatatcgaag gagcctggaa gagaggctac acgggaaaga acattgtggt cactatcctg gatgacggaa ttgagagaac ccatccagat ctgatgcaaa actacgatgc tctggcaagt tgcgacgtga atgggaatga cttggaccca atgcctcgtt atgatgcaag caacgagaac aagcatggga ctcgctgtgc tggagaagtg gcagccgctg caaacaattc gcactgcaca gtcggaattg ctttcaacgc caagatcgga ggagtgcgaa tgctggacgg agatgtcacg gacatggttg aagcaaaatc agttagcttc aacccccagc acgtgcacat ttacagcgcc agctggggcc cggatgatga tggcaagact gtggacggac caggccgcct cacccggcaa gcctttgaaa acggcgttag aatggggcgg agaggcctcg gctctgtgtt tgtttgggca tctggaaatg gtggaaggag caaagaccac tgctcctgtg atggctacac caacagcatc tacaccatct ccatcagcag cactgcagaa agcggaaaga aaccttggta cctggaagag tgttcatcca cgctggccac aacctacagc agcggggagt cctacgataa gaaaatcatc actacagatc tgaggcagcg ttgcacggac aaccacactg ggacgtcagc ctcagccccc atggctgcag gcatcattgc gctggccctg gaagccaatc cgtttctgac ctggagagac gtacagcatg ttattgtcag gacttcccgt gcgggacatt tgaacgctaa tgactggaaa accaatgctg ctggttttaa ggtgagccat ctttatggat ttggactgat ggacgcagaa gccatggtga tggaggcaga gaagtggacc accgttcccc ggcagcacgt gtgtgtggag agcacagacc gacaaatcaa gacaatccgc cctaacagtg cagtgcgctc catctacaaa gcttcaggct gctcggataa ccccaaccgc catgtcaact acctggagca cgtcgttgtg cgcatcacca tcacccaccc caggagagga gacctggcca tctacctgac ctcgccctct ggaactaggt ctcagctttt ggccaacagg ctatttgatc actccatgga aggattcaaa aactgggagt tcatgaccat tcattgctgg ggagaaagag ctgctggtga ctgggtcctt gaagtttatg atactccctc tcagctaagg aactttaaga ctccaggtaa attgaaagaa tggtctttgg tcctctacgg cacctccgtg cagccatatt caccaaccaa tgaatttccg aaagtggaac ggttccgcta tagccgagtt gaagacccca cagacgacta tggcacagag gattatgcag gtccctgcga ccctgagtgc agtgaggttg gctgtgacgg gccaggacca gaccactgca atgactgttt gcactactac tacaagctga aaaacaatac caggatctgt gtctccagct gcccccctgg ccactaccac gccgacaaga agcgctgcag gaagtgtgcc cccaactgtg agtcctqctt tgggagccat ggtgaccaat gcatgtcctg caaatatgga tactttctga atgaagaaac caacagctgt gttactcact gccctgatgg gtcatatcag gataccaaga aaaatctttg ccggaaatgc agtgaaaact gcaagacatg tactgaattc cataactgta cagaatgtag ggatgggtta agcctgcagg gatcccggtg ctctgtctcc tgtgaagatg gacqgtattt caacggccag gactgccagc cctgccaccg cttctgcgcc acttgtgctg gggcaggagc tgatgggtgc attaactgca cagagggcta cttcatggag gatgggagat gcgtgcagag ctgtagtatc agctattact ttgaccactc ttcagagaat ggatacaaat cctgcaaaaa atgtgatatc agttgtttga cgtgcaatgg cccaggattc aagaactgta caagctgccc tagtgggtat ctcttagact taggaatgtg tcaaatggga gccatttgca aggatgcaac ggaagagtcc tgggcggaag gaggcttctg tatgcttgtg aaaaagaaca atctgtgcca acggaaggtt cttcaacaac tttgctgcaa aacatgtaca tttcaaggct ga

In one embodiment, the proprotein convertase used for the present invention comprises an amino acid sequence, which is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 115 to 454 of SEQ ID NO: 18, amino acids 636 to 868 of SEQ ID NO: 18, amino acids 115 to 913 of SEQ ID NO: 18, amino acids 33 to 913 of SEQ ID NO: 18, or SEQ ID NO: 18, wherein the amino acid sequence is capable of cleaving full-length Factor VIII at amino acid residue 1648.

Proprotein convertase subtilisin/kexin type 6 (PCSK6) is also known as paired basic amino acid cleaving enzyme 4 (PACE4), subtilisin/kexin-like protease, subtilisin-like proprotein convertase 4 (SPC4). The gene encoding PCSK6 is known as Pcsk6 or pace4 and has Accession Number M80482.1 in GenBank. The full-length human PCSK6 polypeptide has Accession Number P29122 in UniProtKB/Swiss-Prot entry. Several isoforms produced by alternative splicing are also available as Accession Numbers P29122-1 (Isoform PACE4A-I), P29122-2 (Isoform PACE4A-II), P29122-3 (Isoform PACE4B), P29122-4 (isoform PACE4C), P29122-5 (isoform PACE4CS), P29122-6 (isoform PACE4D), P29122-7 (isoform PACE4E-I), and P29122-8 (isoform PACE4E-II). Isoform PACE4A-I consists of a signal peptide (amino acids 1-63), a propeptide (amino acids 64-149), and active protein domain (amino acids 150-969) including a catalytic domain (amino acids 150-454) and a cysteine-rich motif (amino acids 695-930). The nucleotide and amino acid sequences of isoform PACE4A-I are represented herein as SEQ ID NO: 19 and SEQ ID NO: 20, respectively. Variants of PACE4 include, but are not limited to, the polypeptides with the following mutations: KLK621-623NLD, C502R and T639I. Also included is PCSK6 from a different species, e.g., mouse, rat, monkey, dog, drosophila, or porcine.

PCSK6 Amino Acid Sequence (SEQ ID NO: 20) MPPRAPPAPG PRPPPRAAAA TDTAAGAGGA GGAGGAGGPG FRPLAPRPWR WLLLLALPAA CSAPPPRPVY TNHWAVQVLG GPAEADRVAA AEGYLNLGQI GNLEDYYHEY HSKTFKRSTL SSRGPHTFLR MDPQVKWLQQ QEVKRRVKRQ VRSDPQALYF NDPIWSNMWY LHCGDKNSRC RSEMNVQAAW KRGYTGKNVV VTILDDGIER NHPDLAPNYD SYASYDVNGN DYDPSFRYDA SNENKHGTRC AGEVAASANN SYCIVGIAYN AKIGGIRMLD GDVTDVVEAK SLGIRPNYID IYSASWGPDD DGKTVDGPGR LAKQAFEYGT KKGRQGLGSI FVWASGNGGR EGDYCSCDGY TNSIYTISVS SATENGYKPW YLEECASTLA TTYSSGAEYE RKIVTTDLRQ RCTDGHTGTS VSAPMVAGII ALALEANSQL TWRDVQHLLV KTSRPAELKA SDWKVNGAGH KVSHFYGEGL VDAEALVVEA KKWTAVPSQH MCVAASDKRP RSIPLVQVLR TTALTSACAE HSDQRVVYLE HVVVRTSISH PRRGDLQIYL VSPSGTKSQL LAKRLLDLSN EGFTNWEFMT VHCWSEKAEG QWTLEIQDLP SQVRNPEKQG KLKEWSLILY GTAEHPYHTF SAHQSRSRML ELSAPELEPP KAALSPSQVE VPEDEEDYTA QSTPGSANIL QTSVCHPECG DKCCDGPNAD QCLNCVHFSL GSVKTSRKCV SVCPLGYFGD TAARRCRRCH KGCETCSSRA ATQCLSCRRG FYHHQEMNTC VTLCPAGFYA DESQKNCLKC HPSCKKCVDE PEKCTVCKEC FSLARGSCIP DCEPGTYFDS ELIRCGECHH TCGTCVGPGR EECIHCAKNF HFHDWKCVPA CGEGFYPEEM PGLPHKVCRR CDENCLSCAG SSRNCSRCKT GFTQLGTSCI TNHTCSNADE TFCEMVKSNR LCERKLFIQF CCRTCLLAG PCSK 6 Nucleic Acid Sequence (SEQ ID NO: 19) atgcctccgc gcgcgccgcc tgcgcccggg ccccggccgc cgccccgggc cgccgccgcc accgacaccg ccgcgggcgc ggggggcgcg gggggcgcgg ggggcgccgg cgggcccggg ttccggccgc tcgcgccgcg tccctggcgc tggctgctgc tgctggcgct gcctgccgcc tgctccgcgc ccccgccgcg ccccgtctac accaaccact gggcggtgca agtgctgggc ggcccggccg aggcggaccg cgtggcggcg gcgcacggct acctcaactt gggccagatt ggaaacctgg aagattacta ccatttttat cacagcaaaa cctttaaaag atcaaccttg agtagcagag gccctcacac cttcctcaga atggaccccc aggtgaaatg gctccagcaa caggaagtga aacgaagggt gaagagacag gtgcgaagtg acccgcaggc cctttacttc aacgacccca tttggtccaa catgtggtac ctgcattgtq gcgacaagaa cagtcgctgc cggtcggaaa tgaatgtcca ggcagcgtgg aagagggqct acacaggaaa aaacgtggtg gtcaccatcc ttgatgatqg catagagaga aatcaccctg acctggcccc aaattatgat tcctacgcca gctacgacgt gaacggcaat gattatgacc catctccacg atatgatgcc agcaatgaaa ataaacacgg cactcgttgt gcgggagaag ttgctgcttc agcaaacaat tcctactgca tcgtgggcat agcgtacaat gccaaaatag gaggcatccg catgctggac ggcgatgtca cagatgtggt cgaggcaaag tcgctgggca tcagacccaa ctacatcgac atttacagtg ccagctgggg gccggacgac gacggcaaga cggtggacgg gcccggccga ctggctaagc aggctttcga gtatggcatt aaaaagggcc ggcagggcct gggctccatt ttcgtctggg catctgggaa tggcgggaga gagggggact actgctcgtg cgatggctac accaacagca tctacaccat ctccgtcagc agcgccaccg agaatggcta caagccctgg tacctggaag agtgtgcctc caccctggcc accacctaca gcagtggggc cttttatgag cgaaaaatcg tcaccacgga tctgcgtcag cgctgtaccg atggccacac tgggacctca gtctctgccc ccatggtggc gggcatcatc gccttggctc tagaagcaaa cagccagtta acctggaggg acgtccagca cctgctagtg aagacatccc ggccggccca cctgaaagcg agcgactgga aagtaaacgg cgcgggtcat aaagttagcc atttctatgg atttggtttg gtggacgcag aagctctcgt tgtggaggca aagaagtgga cagcagtgcc atcgcagcac atgtgtgtgg ccgcctcgga caagagaccc aggagcatcc ccttagtgca ggtgctgcgg actacggccc tgaccagcgc ctgcgcggag cactcggacc agcgggtggt ctacttggag cacgtggtgg ttcgcacctc catctcacac ccacgccgag gagacctcca gatctacctg gtttctccct cgggaaccaa gtctcaactt ttggcaaaga ggttgctgga tctttccaat gaagggttta caaactggga attcatgact gtccactgct ggggagaaaa ggctgaaggg cagtggacct tggaaatcca agatctgcca tcccaggtcc gcaacccgga gaagcaaggg aagttgaaag aatggagcct catactgtat ggcacagcag agcacccgta ccacaccttc agtgcccatc agtcccgctc gcggatgctg gagctctcag ccccagagct ggagccaccc aaggctgccc tgtcaccctc ccaggtggaa gttcctgaag atgaggaaga ttacacagct caatccaccc caggctctgc taatatttta cagaccagtg tgtgccatcc ggagtgtggt gacaaaggct gtgatggccc caatgcagac cagtgcttga actgcgtcca cttcagcctg gggagtgtca agaccagcag gaagtgcgtg agtgtgtgcc ccttgggcta ctttggggac acaggagcaa gacgctgtcg ccggtgccac aaggggtgtg agacctgctc cagcagagct gcgacgcagt gcctgtcttg ccgccgcggg ttctatcacc accaggagat gaacacctgt gtgaccctct gtcctgcagg attttatgct gatgaaagtc agaaaaattg ccttaaatgc cacccaagct gtaaaaagtg cgtggatgaa cctgagaaat gtactgtctg taaagaagga ttcagccttg cacggggcag ctgcattcct gactgtgagc caggcaccta ctttgactca gagctgatca gatgtgggga atgccatcac acctgcggaa cctgcgtggg gccaggcaga gaagagtgca ttcactgtgc gaaaaacttc cacttccacg actggaagtg tgtgccagcc tgtggtgagg gcttctaccc agaagagatg ccqggcttgc cccacaaagt gtgtcgaagg tgtgacgaga actgcttgag ctgtgcaggc tccagcagga actgtagcag gtgtaagacg ggcttcacac agctggggac ctcctgcatc accaaccaca cgtgcagcaa cgctgacgag acattctgcg agatggtgaa gtccaaccqg ctgtgcgaac ggaagctctt cattcagttc tgctgccgca cgtgcctcct ggccgggtaa

In one embodiment, the proprotein convertase used for the present invention comprises an amino acid sequence, which is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 150-454 of SEQ ID NO: 20, amino acids 695 to 930 of SEQ ID NO: 20, amino acids 150 to 969 of SEQ ID NO: 20, amino acids 64 to 969 of SEQ ID NO: 20, SEQ ID NO: 20, amino acids 168 to 664 of SEQ ID NO: 20, amino acids 1 to 472 of SEQ ID NO: 20, amino acids 150 to 472 of SEQ ID NO: 20, amino acids 64 to 472 of SEQ ID NO: 20 or any of the amino acid sequences of the isoforms, wherein the amino acid sequence is capable of cleaving full-length Factor VIII at amino acid residue 1648.

Proprotein convertase subtilisin/kexin type 7 (PCSK7) is also known as proprotein convertase 7 (PC7), subtilisin/kexin-like protease PC7, prohormone convertase PC7, hPC8, PC8, subtilisin-like proprotein convertase 7, or lymphoma proprotein convertase. The gene encoding PCSK7 is known as Pcsk7, LPC, PC7, PC8, SPC7 and has Accession Number BT006870.1 in GenBank. The full-length human PCSK7 polypeptide has Accession Number Q16549 in UniProtKB/Swiss-Prot entry and consists of a signal peptide (amino acids 1-37), a propeptide (amino acids 38-141), and the active protein domain (amino acids 142-785) including the catalytic domain (amino acids 142-474). The nucleotide and amino acid sequences of the full-length human PCSK7 are represented herein as SEQ ID NO: 21 and SEQ ID NO: 22, respectively. Variants of PCSK7 include, but are not limited to, the polypeptides with the following mutations: L688V, S689N, R700M, H708Y, R711Q, P52A, A112P, H228R, and A353T. Also included is PCSK7 from a different species, e.g., mouse, rat, monkey, dog, drosophila, or porcine.

PCSK7 Amino Acid Sequence (SEQ ID NO: 22) MPKGRQKVPH LDAPLGLPTC LWEELAGLFL LVEWVMGLAG TGGPDGQGTG GPSWAVHLES LEGDGEEETL EQQADALAQA AGLVNAGRIG ELQGHYLFVQ PAGHRPALEV EAIRQQVEAV LAGHEAVRWH SEQRLLRRAK RSVHFNDPKY PQQWHLNNRR SPGRDINVTG VWERNVTGRG VTVVVVDDGV EHTIQDIAPN YSPEGSYDLN SNDPDPMPHP DVENGNHHGT RCAGEIAAVP NNSFCAVGVA YGSRIAGIRV LDGPLTDSME AVAFNKHYQI NDIYSCSWGP DDDGKTVDGP HQLGKAALQH GVIAGRQGFG SIFVVASONG GQHNDNCNYD GYANSIYTVT IGAVDEEGRM PFYAEECASM LAVTFSGGDK MLRSIVTTDW DLQKGTGCTE GHTGTSAAAP LAAGMIALML QVRPCLTWRD VQHIIVFTAT RYEDRRAEWV TNEAGFSHSH QHGFGLLNAW RLVNAAKIWT SVPYLASYVS PVLKENKAIP QSPRSLEVLW NVSRMDLEMS GDKTLEHVAV TVSITHPRRG SLELKLFCPS GMMSLIGAPR SMDSDPNGFN DWTFSTVRCW GERARGTYRL VIRDVGDESF QVGILRQWQL TLYGSVWSAV DIRDRQRLLE SAMSGKYLHD DFALPCPPGL KIPEEDGYTT TPNTLKTLVL VGCFTVFWTV YYMLEVYLSQ RNVASNQVCR SGPCHWPHRS RKAKEEGTEL ESVPLCSSKD PDEVETESRG PPTTSDLLAP DLLEQGDWSL SQNKSALDCP HQHLDVPHGK EEQIC PCSK 7 Nucleic Acid Sequence (SEQ ID NO: 21) atgccgaagg ggaggcagaa agtgccacac ttggatgccc ccctgggcct gcccacctgc ctctggctgg aattagccgg gctcttctta ctggttccct gggtcatggg cctggcaggg acaggtgggc ctgatggcca gggcacaggg gggccgagct gggctgtgca cctggaaagc ctggaaggtg acggggagga agagactctg gagcagcagg cggatgcctt ggcccaggca gcagggctgg tgaatgctgg acgcatcgga gagcttcagg ggcactacct ctttgtccag cctgctgggc acaggccggc cctggaggtg gaggccatcc ggcagcaggt ggaggctgtg ttggctgggc atgaagctgt gcgctggcac tcagagcaga ggctgctaag gcgggccaag cgcagcgtcc acttcaacga ccccaagtac ccgcagcaat ggcacctgaa taaccgacgg agcccgggca gggacatcaa cgtgacgggt gtgtgggaac gcaatgtgac tgggcgaggg gtgacggtgg tggtagtgga tgacggagtg gaacacacca tccaggacat tgcacccaac tatagccctg agggtagcta tgacctcaac tctaatgacc ctgaccccat gccccacccg gatgtggaga atggcaacca ccatggcacg cgatgtgcag gagagatcgc ggctgtgccc aacaacagct tctgtgccgt gggcgtggcc tacgggagcc gcatcgcagg tatccgggta ctggatggac ctctcacaga cagcatggag gcagtggcgt tcaacaagca ctatcagatc aatgacatct acagctgcag ctggggacca gatgacgatg ggaagacagt ggatggcccc catcagcttg gaaaggctgc cttacaacat ggggtgattg ctggtcgcca gggctttggg agcatctttg tggtagccag tggcaacgga ggccaacaca acgacaactg caactacgat ggctacgcca actccatcta caccgtcacc ataggagctg tggatgagga gggacgcatg cctttctatg cagaagaatg tgcctccatg ctggcagtca ccttcagtgg tggggacaag atgcttcgga gcattgtgac cactgactgg gaccttcaga agggcactgg ctgcactgag ggccacacag ggacctcagc tgcagcgcct ctggcagctg gcatgatagc cttaatgctg caggtgcggc cctgcctcac gtggcgtgac gtccagcaca tcattgtctt cacagccacc cggtatgagg atcgccgtgc agagtgggtc accaacgagg caggcttcag ccatagccac cagcacggtt tcggcctcct caacgcctgg aggctcgtga atgcagccaa gatctggaca tctgtccctt acttagcatc ctacgtcagt cccgtgttaa aagaaaacaa ggcgattccg cagtcccccc gttccctgga ggtcctgtgg aatgtcagca ggatggacct ggagatgtca gggctgaaga ccctggagca tgtggcagtg acagtctcca tcactcaccc acggcgcggc agcttggagc tgaagctgtt ctgccccagt ggcatgatgt ccctcatcgg cgccccccgc agcatggact cggatcccaa cggcttcaat gactggacct tctccactgt gcgatgctgg ggggagagag cccgagggac ctacaggctt gtcatcaggg atgtcgggga tgagtcattc caggtcggca tcctccggca atggcagctg accctatatg gctctgtgtg gagtgcagta gacatcaggg acagacaaag gctgttagag agtgccatga gtggaaaata cctgcacgat gacttcgccc tgccctgccc accggggctg aaaattcctg aggaagatgg ttacaccatc accgccaaca ccctcaagac cctggtgctg gtaggctgtt tcaccgtctt ctggactgtt tactacatgc tggaagtata tttgagccag aggaatgtgg cttccaatca agtttgtagg agtggaccct gccactggcc ccatcggagc cggaaagcca aggaggaagg gacagagcta gaatcagtgc cactttgcag cagcaaggat ccagacgaag tggaaacaga gagcaggggc cctcccacca cctctgacct ccttgcccca gacctgctgg agcaagggga ctggagcctg tcccagaaca agagcgccct ggactgccct catcagcacc tagacgtacc gcacgggaag gaggagcaga tctgctag

In another embodiment, the proprotein convertase used for the present invention comprises an amino acid sequence, which is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 142 to 474 of SEQ ID NO: 22, amino acids 14 to 785 of SEQ ID NO: 22, amino acids 38 to 785 of SEQ ID NO: 22, or SEQ ID NO: 22, wherein the amino acid sequence is capable of cleaving full-length Factor VIII at amino acid residue 1648.

Proprotein convertase subtilisin/kexin type 8 (PCSK8) is also known as membrane-bound transcription factor peptidase, site 1 (MBTPS1), S1P endopeptidase, subtilisin/kexin-isozyme 1 (SKI-1), and site 1 protease (S1P). The gene encoding Pcsk8 is known as MBTPS1 and has Accession Number BC114555.1 in GenBank. The full-length human PCSK8 polypeptide has Accession Number Q14703 in UniProtKB/Swiss-Prot entry and consists of the signal peptide (amino acids 1-17), the propeptide (amino acids 18-186), and the active protein domain (amino acids 187-1052) including the serine protease domain (amino acids 218-414). The nucleotide and amino acid sequences of the full-length human PCSK8 are represented herein as SEQ ID NO: 23 and SEQ ID NO: 24, respectively. Variants of PCSK8 include, but are not limited to, the polypeptides with the following mutations: I6T and R90G. Also included is PCSK8 from a different species, e.g., mouse, rat, monkey, dog, drosophila, or porcine.

PCSK8 Amino Acid Sequence (SEQ ID NO: 24) MKLVNIWLLL LVVLLCGKKH LGDRLEKKSF EKAPCPGCSH LTLKVEFSST VVEYEYIVAF NGYFTAKARN SFISSALKSS EVDNWRIIPR NNPSSDYPSD FEVIQIKEKQ KAGLLTLEDH PNIKRVTPQR KVFRSLKYAE SDPTVPCNET RWSQKWQSSR PLRRASLSLC SCFWHATGRH SSRRLLRAIP RQVAQTLQAD VLWQMGYTGA NVRVAVFDTG LSEKHPHFKN VKERTNWTNE RTLDDGLGHG TFVAGVIASM RECQGFAPDA ELHIERVETN NQVSYTSWFL DAFNYAILKK IDVLNLSIGG PDFMDHPFVD KVWELTANNV IMVSAIGNDG PLYGTLNNPA DQMDVIGVGG IDFEDNIARF SSRGMTTWEL PGGYGRMKPD IVTYGAGVRG SGVKGGCRAL SGTSVASPVV AGAVTLLVST VQKRELVNPA SMKQALIASA RRLPGVNMFE QGHGKLDLLR AYQILNSYKP QASLSPSYID LTECPYMWPY CSQPIYYGGM PTVVNVTILN GMGVTGRIVD KPDWQFYLPQ NGDNIEVAPS YSSVLWPWSG YLAISISVTK KAASWEGIAQ GHVMITVASP AETESKNGRF QTSTVKLPIK VKIIPTPPRS KRVLWDQYEN LRYPPGYFPR DNLRMKNDPL DWNGDHIHTN FRDMYQHLRS MGYFVEVLGA PFTCFDASQY GTLLMVDSEE EYFPEETAKL RRDVDNCLSL VIFSDWYNTS VMRKVKFYDE NTRQWWMPDT GGANIPALNE LLSVWNMGFS DGLYEGEFTL ANHDMYYASC CSIAKFPEDG VVITQTFKDQ GLEVLKQETA VVENVFILGL YQIPAEGGGR TVLYGDSNCL DDSHRQKDCF WLLDALLQYT SYGVTPPSLS HSGNRQRPPS CAGSVTPERM EGNHLHRYSK VLEAHLGDPK PRPLPACPRL SWAKPQPLNE TAPSNLWKHQ KLLSIDLDKV VLPNFRSNRP QVRPLSPGES GAWDIPGGIM PGRYNQEVGQ TIPVFAFLGA MVVLAFFVVQ INKAKSRPHR RKPRVKRPQL MQQVHPPKTP SV PCSK 8 Nucleic Acid Sequence (SEQ ID NO: 23) atgaagcttg tcaacatctg gctgcttctg ctcgtggttt tgctctgtgg gaagaaacat ctgggcgaca gactggaaaa gaaatctttt gaaaaggccc catgccctgg ctgttcccac ctgactttga aggtggaatt ctcatcaaca gttgtggaat atgaatatat tgtggctttc aatggatact ttacagccaa agctagaaat tcatttattt caagtgccct gaagaggagt gaagtagaca attggagaat tatacctcga aacaatccat ccagtgacta ccctagtgat tttgaggtga ttcagataaa agaaaaacag aaagcggggc tgctaacact tgaagatcat ccaaacatca aacgggtcac gccccaacga aaagtctttc gttccctcaa gtatgctgaa tctgacccca cagtaccctg caatgaaacc cggtggagcc agaagtggca atcatcacgt cccctgcgaa gagccagcct ctccctgggc tctggcttct ggcatgctac gggaaggcat tcgagcagac ggctgctgag agccatcccg cgccaggttg cccagacact gcaggcagat gtgctctggc agatgggata tacaggtgct aatgtaagag ttgctgtttt tgacactggg ctgagcgaga agcatcccca cttcaaaaat gtgaaggaga gaaccaactg gaccaacgag cgaacgctgg acgatgggtt gggccatggc acattcgtgg caggtgtgat agccagcatg agggagtgcc aaggatttgc tccagatgca gaacttcaca ttttcagggt ctttaccaat aatcaggtat cttacacatc ttggtttttg gacgccttca actatgccat tttaaagaag atcgacgtgt taaacctcag catcggcggc ccggacttca tggatcatcc gtttgttgac aaggtgtggg aattaacagc taacaatgta atcatggttt ctgctattgg caatgacgga cctctttatg gcactctgaa taaccctgct gatcaaatgg atgtgattgg agtaggcggc attgactttg aagataacat cgcccgcttt tcttcaaggg gaatgactac ctgggagcta ccaggaggct acggtcgcat gaaacctgac attgtcacct atggtgctgg cgtgcggggt tctggcgtga aaggggggtg ccgggccctc tcagggacca gtgttgcttc tccagtggtt gcaggtgctg tcaccttgtt agtgagcaca gtccagaagc gtgagctggt gaatcccgcc agtatgaagc aggccctgat cgcgtcagcc cggaggctcc ccggggtcaa catgtttgag caaggccacg gcaagctcga tctgctcaga gcctatcaga tcctcaacag ctacaagcca caggcaagtt tgagccccag ctacatagat ctgactgagt gtccctacat gtggccctac tgctcccagc ccatctacta tggaggaatg ccgacagttg ttaatgtcac catcctcaac ggcatgggag tcacaggaag aattgtagat aagcctgact ggcagcccta tttgccacag aacggagaca acattgaagt tgccttctcc tactcctcgg tcttatggcc ttggtcgggc tacctggcca tctccatttc tgtgaccaag aaagcggctt cctgggaagg cattgctcag ggccatgtca tgatcactgt ggcttcccca gcagagacag agtcaaaaaa tggtgcagaa cagacttcaa cagtaaagct ccccattaag gtgaagataa ttcctactcc cccgcgaagc aagagagttc tctgggatca gtaccacaac ctccgctatc cacctggcta tttccccagg gataatttaa ggatgaagaa tgacccttta gactggaatg gtgatcacat ccacaccaat ttcagggata tgtaccagca tctgagaagc atgggctact ttgtagaggt cctcggggcc cccttcacgt gttttgatgc cagtcagtat ggcactttgc tgatggtgga cagtgaggag gagtacttcc ctgaagagat cgccaagctc cggagggacg tggacaacgg cctctcgctc gtcatcttca gtgactggta caacacttct gttatgagaa aagtgaagtt ttatgatgaa aacacaaggc agtggtggat gccggatacc ggaggagcta acatcccagc tctgaatgag ctgctgtctg tgtggaacat ggggttcagc gatggcctgt atgaagggga gttcaccctg gccaaccatg acatgtatta tgcgtcaggg tgcagcatcg cgaagtttcc agaagatggc gtcgtgataa cacagacttt caaggaccaa ggattggagg ttttaaagca ggaaacagca gttgttgaaa acgtccccat tttgggactt tatcagattc cagctgaggg tggaggccgg attgtactgt atggggactc caattgcttg gatgacagtc accgacagaa ggactgcttt tggcttctgg atgccctcct ccagtacaca tcgtatgggg tgacaccgcc tagcctcagt cactctggga accgccagcg ccctcccagt ggagcaggct cagtcactcc agagaggatg gaaggaaacc atcttcatcg gtactccaag gttctggagg cccatttggg agacccaaaa cctcggcctc taccagcctg tccacgcttg tcttgggcca agccacagcc tttaaacgag acggcgccca gtaacctttg gaaacatcag aagctactct ccattgacct ggacaaggtg gtgttaccca actttcgatc gaatcgccct caagtgaggc ccttgtcccc tggagagagc ggcgcctggg acattcctgg agggatcatg cctggccgct acaaccagga ggtgggccag accattcctg tctttgcctt cctgggagcc atggtggtcc tggccttctt tgtggtacaa atcaacaagg ccaagagcag gccgaagcgg aggaagccca gggtgaagcg cccgcagctc atgcagcagg ttcacccgcc aaagacccct tcggtgtga

In one embodiment, the proprotein convertase used for the present invention comprises an amino acid sequence, which is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 218 to 414 of SEQ ID NO: 24, amino acids 187 to 1052 of SEQ ID NO: 24, amino acids 18 to 1052 of SEQ ID NO: 24, or SEQ ID NO: 24, wherein the amino acid sequence is capable of cleaving full-length Factor VIII at amino acid residue 1648.

Proprotein convertase subtilisin/kexin type 9 (PCSK9) is also known as proprotein convertase 9 (PC9), subtilisin/kexin-like protease PC9, or neural apoptosis-regulated convertase 1 (NARC-1). The gene encoding Pcsk9 is known as psec0052, narc-1, and pc9 and has Accession Number AX127530 in GenBank. The full-length human PCSK9 polypeptide has Accession Number Q8NBP7 in UniProtKB/Swiss-Prot entry and consists of the signal peptide (amino acids 1-30), the propeptide (amino acids 31-152), and the active protein domain (amino acids 153-692) including the peptidase S8 domain (amino acids 161-431). The nucleotide and amino acid sequences of the full-length human PCSK8 are represented herein as SEQ ID NO: 25 and SEQ ID NO: 26, respectively. Variants of PCSK9 include, but are not limited to, the polypeptides with the following mutations: L23LL, R46L, A53V, E57K, T77I, R93C, G106R, V114A, S127R, D129G, N157K, R215H, F216L, R218S, Q219E, R237W, A239D, L253F, R357H, D374H, D374Y, H391N, H1417Q, N425S, A443T, G452D, R469W, I474V, E482G, R496W, F515L, A522T, H553R, Q554E, P616L, Q619P, S668R, E670G, C67A, H226A, N533A, or V423A. Also included is PCSK9 from a different species, e.g., mouse, rat, monkey, dog, drosophila, or porcine.

PCSK9 Amino Acid Sequence (SEQ ID NO: 26) MGTVSSRRSW WPLPLLLLLL LLLGPAGARA QEDEDGDYEE LVLALRSEED GLAEAPEHGT TATFHRCAKD PWRLPGTYVV VLKEETHLSQ SERTARRLQA QAARRGYLTK ILHVFHGLLP GFLVKMSGDL LELALKLPHV DYIEEDSSVE AQSIPWNLER ITPPRYRADE YQPPDGGSLV EVYLLDTSIQ SDHREIEGRV MVTDFENVPE EDGTREHRQA SKCDSHGTHL AGVVSGRDAG VAKGASMRSL RVLNCQGKGT VSGILIGLEF TRKSQLVQPV GPLVVLLPLA GGYSRVLNAA CQRLARAGVV LVTAAGNERD DACLYSPASA PEVITVGATN AQDQPVTLGT LGTNFGRCVD LEAPGEDIIG ASSDCSTCEV SQSGTSQAAA HVAGIAAMML SAEPELTLAE LRORLIEFSA KDVINEAWFP EDQRVLTPNL VAALPPSTHG AGWOLFCRTV WSAHSGPTRM ATAIARCAPD EELLSCSSFS RSGKRRGERM EAQGGKLVCR AHNAFGGEGV YAIARCCLLP QANCSVHTAP PAEASMGTRV HCHQQGHVLT GCSSHWEVED LGTHKPPVLR PRGQPNQCVG HREASIHASC CHAPGLECKV KEHGIPAPQE QVTVACEEGW TLTGCSALPG TSHVLGAYAV DNTCVVRSRD VSTTGSTSEE AVTAVAICCR SRHLAQASQE LQ PCSK9 Nucleic Acid Sequence (SEQ ID NO: 25) atgggcaccg tcagctccag gcggtcctgg tggccgctgc cactgctgct gctgctgctg ctgctcctgg gtcccgcggg cgcccgtgcg caggaggacg aggacggcga ctacgaggag ctggtgctag ccttgcgttc cgaggaggac ggcctggccg aagcacccga gcacggaacc acagccacct tccaccgctg cgccaaggat ccgtggaggt tgcctggcac ctacgtggtg gtgctgaagg aggagaccca cctctcgcag tcagagcgca ctgcccgccg cctgcaggcc caggctgccc gccggggata cctcaccaag atcctgcatg tcttccatgg ccttcttcct ggcttcctgg tgaagatgag tggcgacctg ctggagctgg ccttgaagtt gccccatgtc gactacatcg aggaggactc ctctgtcttt gcccagagca tcccgtggaa cctggagcgg attacccctc cacggtaccg ggcggatgaa taccagcccc ccgacggagg cagcctggtg gaggtgtatc tcctagacac cagcatacag agtgaccacc gggaaatcga gggcagggtc atggtcaccg acttcgagaa tgtgcccgag gaggacggga cccgcttcca cagacaggcc agcaagtgtg acagtcatgg cacccacctg gcaggggtgg tcagcggccg ggatgccggc gtggccaagg gtgccagcat gcgcagcctg cgcgtgctca actgccaagg gaagggcacg gttagcggca ccctcatagg cctggagttt attcggaaaa gccagctggt ccagcctgtg gggccactgg tggtgctgct gcccctggcg ggtgggtaca gccgcgtcct caacgccgcc tgccagcgcc tggcgagggc tggggtcgtg ctggtcaccg ctgccggcaa cttccgggac gatgcctgcc tctactcccc agcctcagct cccgaggtca tcacagttgg ggccaccaat gcccaggacc agccggtgac cctggggact ttggggacca actttggccg ctgtgtggac ctctttgccc caggggagga catcattggt gcctccagcg actgcagcac ctgctttgtg tcacagagtg ggacatcaca ggctgctgcc cacgtggctg gcattgcagc catgatgctg tctgccgagc cggagctcac cctggccgag ttgaggcaga gactgatcca cttctctgcc aaagatgtca tcaatgaggc ctggttccct gaggaccagc gggtactgac ccccaacctg gtggccgccc tgccccccag cacccatggg gcaggttggc agctgttttg caggactgtg tggtcagcac actcggggcc tacacggatg gccacagcca tcgcccgctg cgccccagat gaggagctgc tgagctgctc cagtttctcc aggagtggga agcggcgggg cgagcgcatg gaggcccaag ggggcaagct ggtctgccgg gcccacaacg cttttggggg tgagggtgtc tacgccattg ccaggtgctg cctgctaccc caggccaact gcagcgtcca cacagctcca ccagctgagg ccagcatggg gacccgtgtc cactgccacc aacagggcca cgtcctcaca ggctgcagct cccactggga ggtggaggac cttggcaccc acaagccgcc tgtgctgagg ccacgaggtc agcccaacca gtgcgtgggc cacagggagg ccagcatcca cgcttcctgc tgccatgccc caggtctgga atgcaaagtc aaggagcatg gaatcccggc ccctcaggag caggtgaccg tggcctgcga ggagggctgg accctgactg gctgcagtgc cctccctggg acctcccacg tcctgggggc ctacgccgta gacaacacgt gtgtagtcag gagccgggac gtcagcacta caggcagcac cagcgaagag gccgtgacag ccgttgccat ctgctgccgg agccggcacc tggcgcaggc ctcccaggag ctccagtga

In one embodiment, the proprotein convertase used for the present invention comprises an amino acid sequence at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 161 to 431 of SEQ ID NO: 26, amino acids 153 to 692 of SEQ ID NO: 26, amino acids 31 to 692 of SEQ ID NO: 26, or SEQ ID NO: 26, wherein the amino acid sequence is capable of cleaving full-length Factor VIII at amino acid residue 1648.

The proprotein convertase can comprise a yeast Kex2 protease, which is also known as Kexin or proteinase YSCF. The gene encoding the Kex2 protease is known as kex2 or qds1 and has Accession Number AAA3478.1 in GenBank. The full-length yeast Kex2 protease has Accession Number P13134 in UniProtKB/Swiss-Prot entry and comprises the signal peptide (amino acids 1-19), the propeptide (amino acids 20-113), and the active protein domain (amino acids 114-814) including the catalytic domain (amino acids 114-461). The nucleotide and amino acid sequences of the full-length yeast Kex2 protease are represented herein as SEQ ID NO: 27 and SEQ 113 NO: 28, respectively.

Yeast Kex2 amino acid sequence (SEQ ID NO: 28) MKVRKYITLC FWWAFSTSAL VSSQQIPLKD HTSRQYFAVE SNETISRIEE MHPNWKYEHD VRGLPNHYVF SKELLKLGKR SSLEELQGDN NDHILSVHDL FPRNDLFKRL PVPAPPMDSS LLPVKEAEDK LSINDPLFER QWHLVNPSFP GSDINVLDLW YNNITGAGVV AAIVDDGLDY ENEDLKONFC AEGSWDFNDN TNLPKPRLSD DYHGTRCAGE IAAKKGNNFC GVGVGYNAKI SGIRILSGDI TTEDEAASLI YGLDVNDIYS CSWGPADDGR HLQGPSDLVK KALVKGVIEG RDSKGAIYVF ASGNGGTRGD NCNYDGYINS IYSITIGAID HKDLEPPYSE GCSAVMAVTY SSGSGEYIHS SDINGRCSNS HGGTSAAAPL AAGVYTLLLE ANPNLIWRDV QYLSILSAVG LEKNADGDWR DSAMGKKYSH RYGFGKIDAH KLIEMSKTWE NVNAQTWFYL PTLYVSQSTN STEETLESVI TISEKSLQDA NEKRIEHVIV TVDIDTEIRG ITTVDLISPA GIISNLGVVR PRDVSSEGFK DWTFMSVAHW GENGVGDWKI KVKTTENGHR IDFHSWRLKL PGESIDSSKT ETFVFGNDKE EVEPAATEST VSQYSASSTS ISISATSTSS ISIGVETSAI PQTTTASTDP DSDPNTPKKL SSPRQAMHYF LTIFLIGATE LVLYFMFFMK SRRRIRRSRA ETYEFDIIDI DSEYDSTLDN GTSGITEPEE VEDFDFDLSD EDHLASLSSS ENGDAEHTID SVLTNENPFS DPIKQKFPND ANAESASNKL QELQPDVPPS SGRS Yeast Kex 2 Nucleic Acid Sequence (SEQ ID NO: 27) atgaaagtga ggaaatatat tactttatgc ttttggtggg ccttttcaac atccgctctt gtatcatcac aacaaattcc attgaaggac catacgtcac gacagtattt tgctgtagaa agcaatgaaa cattatcccg cttggaggaa atgcatccaa attggaaata tgaacatgat gttcgagggc taccaaacca ttatgttttt tcaaaagagt tgctaaaatt gggcaaaaga tcatcattag aagagttaca gggggataac aacgaccaca tattatctgt ccatgattta ttcccgcgta acgacctatt taagagacta ccggtgcctg ctccaccaat ggactcaagc ttgttaccgg taaaagaagc tgaggataaa ctcagcataa atgatccgct ttttgagagg cagtggcact tggtcaatcc aagttttcct ggcagtgata taaatgttct tgatctgtgg tacaataata ttacaggcgc aggggtcgtg gctgccattg ttgatgatgg ccttgactac gaaaatgaag acttgaagga taatttttgc gctgaaggtt cttgggattt caacgacaat accaatttac ctaaaccaag attatctgat gactaccatg gtacgagatg tgcaggtgaa atagctgcca aaaaaggtaa caatttttgc ggtgtcgggg taggttacaa cgctaaaatc tcaggcataa gaatcttatc cggtgatatc actacggaag atgaagctgc gtccttgatt tatggtctag acgtaaacga tatatattca tgctcatggg gtcccgctga tgacggaaga catttacaag gccctagtga cctggtgaaa aaggctttag taaaaggtgt tactgaggga agagattcca aaggagcgat ttacgttttt gccagtggaa atggtggaac tcgtggtgat aattgcaatt acgacggcta tactaattcc atatattcta ttactattgg ggctattgat cacaaagatc tacatcctcc ttattccgaa ggttgttccg ccgtcatggc agtcacgtat tcttcaggtt caggcgaata tattcattcg agtgatatca acggcagatg cagtaatagc cacggtggaa cgtctgcggc tgctccatta gctgccggtg tttacacttt gttactagaa gccaacccaa acctaacttg gagagacgta cagtatttat caatcttgtc tgcggtaggg ttagaaaaga acgctgacgg agattggaga gatagcgcca tggggaagaa atactctcat cgctatggct ttggtaaaat cgatgcccat aagttaattg aaatgtccaa gacctgggag aatgttaacg cacaaacctg gttttacctg ccaacattgt atgtttccca gtccacaaac tccacggaag agacattaga atccgtcata accatatcag aaaaaagtct tcaagatgct aacttcaaga gaattgagca cgtcacggta actgtagata ttgatacaga aattagggga actacgactg tcgatttaat atcaccagcg gggataattt caaacuttgg cgttgtaaga ccaagagatg tttcatcaga gggattcaaa gactggacat tcatgtctgt agcacattgg ggtgagaacg gcgtaggtga ttggaaaatc aaggttaaga caacagaaaa tggacacagg attgacttcc acagttggag gctgaagctc tttggggaat ccattgattc atctaaaaca gaaactttcg tctttggaaa cgataaagag gaggttgaac cagctgctac agaaagtacc gtatcacaat attctgccag ttcaacttct atttccatca gcgctacttc tacatcttct atctcaattg gtgtggaaac gtcggccatt ccccaaacga ctactgcgag taccgatcct gattctgatc caaacactcc taaaaaactt tcctctccta ggcaagccat gcattatttt ttaacaatat ttttgattgg cgccacattt ttggtgttat acttcatgtt ttttatgaaa tcaaggagaa ggatcagaag gtcaagagcg gaaacgtatg aattcgatat cattgataca gactctgagt acgattctac tttggacaat ggaacttccg gaattactga gcccgaagag gttgaggact tcgattttga tttgtccgat gaagaccatc ttqcaagttt gtcttcatca gaaaacggtg atgctgaaca tacaattgat agtgtactaa caaacgaaaa tccatttagt gaccctataa agcaaaagtt cccaaatgac gccaacgcag aatctgcttc caataaatta caagaattac agcctgatgt tcctccatct tccggacgat cgtga

A proprotein convertase can comprise an amino acid sequence at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to amino acids 114-461 of SEQ ID NO: 28, amino acids 153 to 814 of SEQ ID NO: 28, or SEQ ID NO: 28, wherein the amino acid sequence is capable of cleaving full-length Factor VIII at amino acid residue 1648.

In certain embodiments, the proprotein convertase used in this invention is selected from the group consisting of PCSK3, PCSK5, PCSK7, yeast Kex2, and a combination thereof. In some embodiments, the proprotein convertase is selected from the group consisting of PCSK5, PCSK7, and both.

In other embodiments, the proprotein convertase for this invention is selected from the group consisting of PCSK1, PCSK2, PCSK4, PCSK6, PCSK8, PCSK9, and a combination thereof.

In certain embodiments, the present invention is directed to a method of decreasing nonprocessed Factor VIII comprising (a) expressing Factor VIII from a polynucleotide in a host cell, wherein the endogenous processing enzymes of the host cell are insufficient to convert all of the Factor VIII to its processed form, (b) adding a proprotein convertase to the host cell, and (c) decreasing the nonprocessed Factor VIII by processing with the proprotein convertase.

In other embodiments, the present invention includes a method of decreasing nonprocessed Factor VIII comprising (a) expressing Factor VIII from a first polynucleotide in a first host cell, wherein the endogenous processing enzymes of the host cell are insufficient to convert all of the Factor VIII to its processed form, (b) expressing a proprotein convertase from a second polynucleotide in a second host cell, and (c) decreasing the nonprocessed Factor VIII by culturing the first host cell and the second host cell together, wherein the proprotein convertase expressed in the second host cell decreases the nonprocessed Factor VIII expressed in the first host cell.

Vector and Host Cells

When a proprotein convertase and an nonprocessed Factor VIII are to be coexpressed within a cell, in one embodiment, the cell contains a first nucleotide sequence encoding an nonprocessed Factor VIII or a fusion protein thereof, and a second nucleotide sequence encoding a functional protein convertase polypeptide. The first nucleotide sequence and the second nucleotide sequence can be in the same expression vector or different expression vector.

As used herein, an expression vector refers to any nucleic acid construct which contains the necessary elements for the transcription and translation of an inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation, when introduced into an appropriate host cell. Expression vectors can include plasmids, phagemids, viruses, and derivatives thereof.

Expression vectors of the invention will include polynucleotides encoding a protein convertase and Factor VIII.

In one embodiment, a coding sequence for Factor VIII or proprotein convertase is operably linked to an expression control sequence. As used herein, two nucleic acid sequences are operably linked when they are covalently linked in such a way as to permit each component nucleic acid sequence to retain its functionality. A coding sequence and a gene expression control sequence are said to be operably linked when they are covalently linked in such a way as to place the expression or transcription and/or translation of the coding sequence under the influence or control of the gene expression control sequence. Two DNA sequences are said to be operably linked if induction of a promoter in the 5′ gene expression sequence results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequence, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a gene expression sequence would be operably linked to a coding nucleic acid sequence if the gene expression sequence were capable of effecting transcription of that coding nucleic acid sequence such that the resulting transcript is translated into the desired protein or polypeptide.

A gene expression control sequence as used herein is any regulatory nucleotide sequence, such as a promoter sequence or promoter-enhancer combination, which facilitates the efficient transcription and translation of the coding nucleic acid to which it is operably linked. The gene expression control sequence may, for example, be a mammalian or viral promoter, such as a constitutive or inducible promoter. Constitutive mammalian promoters include, but are not limited to, the promoters for the following genes: hypoxanthine phosphoribosyl transferase (HPRT), adenosine deaminase, pyruvate kinase, beta-actin promoter, and other constitutive promoters. Exemplary viral promoters which function constitutively in eukaryotic cells include, for example, promoters from the cytomegalovirus (CMV), simian virus (e.g., SV40), papilloma virus, adenovirus, human immunodeficiency virus (HIV), Rous sarcoma virus, cytomegalovirus, the long terminal repeats (LTR) of Moloney leukemia virus, and other retroviruses, and the thymidine kinase promoter of herpes simplex virus. Other constitutive promoters are known to those of ordinary skill in the art. The promoters useful as gene expression sequences of the invention also include inducible promoters. Inducible promoters are expressed in the presence of an inducing agent. For example, the metallothionein promoter is induced to promote transcription and translation in the presence of certain metal ions. Other inducible promoters are known to those of ordinary skill in the art.

In general, the gene expression control sequence shall include, as necessary, 5′ non-transcribing and 5′ non-translating sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5′ non-transcribing sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined coding nucleic acid. The gene expression sequences optionally include enhancer sequences or upstream activator sequences as desired.

Viral vectors include, but are not limited to, nucleic acid sequences from the following viruses: retrovirus, such as Moloney murine leukemia virus, Harvey murine sarcoma virus, murine mammary tumor virus, and Rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyomaviruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known in the art. Certain viral vectors are based on non-cytopathic eukaryotic viruses in which non-essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses, the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell line with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, M., Gene Transfer and Expression, A Laboratory Manual, W.H. Freeman Co., New York (1990) and Murry, E. J., Methods in Molecular Biology, Vol. 7, Humana Press, Inc., Cliffton, N.J. (1991).

In one embodiment, the virus is an adeno-associated virus, a double-stranded DNA virus. The adeno-associated virus can be engineered to be replication-deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well-known to those of skill in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been found to be particularly advantageous for delivering genes to cells in vivo because of their inability to replicate within and integrate into a host genome. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operably encoded within the plasmid. Some commonly used plasmids available from commercial suppliers include pBR322, pUC18, pUC19, various pcDNA plasmids, pRC/CMV, various pCMV plasmids, pSV40, and pBlueScript. Additional examples of specific plasmids include pcDNA3.1, catalog number V79020; pcDNA3.1/hygro, catalog number V87020; pcDNA4/myc-His, catalog number V86320; and pBudCE4.1, catalog number V53220, all from Invitrogen (Carlsbad, Calif.). Other plasmids are well-known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using standard molecular biology techniques to remove and/or add specific fragments of DNA.

In one insect expression system that may be used to produce the proteins of the invention, Autographa californica nuclear polyhidrosis virus (AcNPV) is used as a vector to express the foreign genes. The virus grows in Spodoptera frugiperda cells. A coding sequence may be cloned into non-essential regions (for example, the polyhedron gene) of the virus and placed under control of an ACNPV promoter (for example, the polyhedron promoter). Successful insertion of a coding sequence will result in inactivation of the polyhedron gene and production of non-occluded recombinant virus (i.e., virus lacking the proteinaceous coat coded for by the polyhedron gene). These recombinant viruses are then used to infect Spodoptera frugiperda cells in which the inserted gene is expressed. (see, e.g., Smith et al. (1983) J Viral 46:584; U.S. Pat. No. 4,215,051). Further examples of this expression system may be found in Ausubel et al., eds. (1989) Current Protocols in Molecular Biology, Vol. 2, Greene Publish. Assoc. & Wiley Interscience.

Another system which can be used to express the proteins of the invention is the glutamine synthetase gene expression system, also referred to as the “GS expression system” (Lonza Biologics PLC, Berkshire UK). This expression system is described in detail in U.S. Pat. No. 5,981,216.

In mammalian host cells, a number of viral based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, a coding sequence may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing peptide in infected hosts. See, e.g., Logan & Shenk (1984) Proc Natl Acad Sci USA 81:3655). Alternatively, the vaccinia 7.5 K promoter may be used. See, e.g., Mackett et al. (1982) Proc Natl Acad Sci USA 79:7415; Mackett et al. (1984) J Virol 49:857; Panicali et al. (1982) Proc Natl Acad Sci USA 79:4927.

To increase efficiency of production, the polynucleotides can be designed to encode multiple units of the protein of the invention separated by enzymatic cleavage sites. The resulting polypeptide can be cleaved (e.g., by treatment with the appropriate enzyme) in order to recover the polypeptide units. This can increase the yield of polypeptides driven by a single promoter. When used in appropriate viral expression systems, the translation of each polypeptide encoded by the mRNA is directed internally in the transcript; e.g., by an internal ribosome entry site, IRES. Thus, the polycistronic construct directs the transcription of a single, large polycistronic mRNA which, in turn, directs the translation of multiple, individual polypeptides. This approach eliminates the production and enzymatic processing of polyproteins and may significantly increase yield of polypeptide driven by a single promoter.

Vectors used in transformation will usually contain a selectable marker used to identify transformants. In bacterial systems, this can include an antibiotic resistance gene such as ampicillin or kanamycin. Selectable markers for use in cultured mammalian cells include genes that confer resistance to drugs, such as neomycin, hygromycin, and methotrexate. The selectable marker may be an amplifiable selectable marker. One amplifiable selectable marker is the dihydrofolate reductase (DHFR) gene. Simonsen C C et al. (1983) Proc Natl Acad Sci USA 80:2495-9. Selectable markers are reviewed by Thilly (1986) Mammalian Cell Technology, Butterworth Publishers, Stoneham, Mass., and the choice of selectable markers is well within the level of ordinary skill in the art.

Selectable markers may be introduced into the cell on a separate plasmid at the same time as the gene of interest, or they may be introduced on the same plasmid. If on the same plasmid, the selectable marker and the gene of interest may be under the control of different promoters or the same promoter, the latter arrangement producing a dicistronic message. Constructs of this type are known in the art (for example, U.S. Pat. No. 4,713,339).

The expression vectors can encode for tags that permit for easy purification of the recombinantly produced protein. Examples include, but are not limited to vector pUR278 (Ruther et al. (1983) EMBO J 2:1791) in which coding sequences for the protein to be expressed may be ligated into the vector in frame with the lac z coding region so that a tagged fusion protein is produced; pGEX vectors may be used to express proteins of the invention with a glutathione S-transferase (GST) tag. These proteins are usually soluble and can easily be purified from cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione. The vectors include cleavage sites (thrombin or Factor Xa protease or PRESCISSION PROTEASE™ (Pharmacia, Peapack, N.J.)) for easy removal of the tag after purification.

The expression vector or vectors are then transfected or co-transfected into a suitable target cell, which will express the polypeptides. Transfection techniques known in the art include, but are not limited to, calcium phosphate precipitation (Wigler et al. (1978) Cell 14:725), electroporation (Neumann et al. (1982) EMBO J 1:841), and liposome-based reagents. A variety of host-expression vector systems may be utilized to express the proteins described herein including both prokaryotic and eukaryotic cells. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli) transformed with recombinant bacteriophage DNA or plasmid DNA expression vectors containing an appropriate coding sequence; yeast or filamentous fungi transformed with recombinant yeast or fungi expression vectors containing an appropriate coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an appropriate coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus or tobacco mosaic virus) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an appropriate coding sequence; or animal cell systems, including mammalian cells (e.g., HEK 293, CHO, Cos, HeLa. HKB11, and BHK cells).

In one embodiment, the host cell is a eukaryotic cell. As used herein, a eukaryotic cell refers to any animal or plant cell having a definitive nucleus. Eukaryotic cells of animals include cells of vertebrates, e.g., mammals, and cells of invertebrates, e.g., insects. Eukaryotic cells of plants specifically can include, without limitation, yeast cells. A eukaryotic cell is distinct from a prokaryotic cell, e.g., bacteria.

In certain embodiments, the eukaryotic cell is a mammalian cell. A mammalian cell is any cell derived from a mammal. Mammalian cells specifically include, but are not limited to, mammalian cell lines. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a HEK 293 cell, which is a human embryonic kidney cell line. HEK 293 cells are available as CRL-1533 from American Type Culture Collection, Manassas, Va., and as 293-H cells, Catalog No. 11631-017 or 293-F cells, Catalog No. 11625-019 from Invitrogen (Carlsbad, Calif.). In some embodiments, the mammalian cell is a PER.C6® cell, which is a human cell line derived from retina. PER.C6® cells are available from Crucell (Leiden, The Netherlands). In other embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. CHO cells are available from American Type Culture Collection, Manassas, Va. (e.g., CHO-K1; CCL-61). In still other embodiments, the mammalian cell is a baby hamster kidney (BHK) cell. BHK cells are available from American Type Culture Collection, Manassas, Va. (e.g., CRL-1632). In some embodiments, the mammalian cell is a HKB11 cell, which is a hybrid cell line of a HEK293 cell and a human B cell line. Mei et al., Mol. Biotechnol. 34(2): 165-78 (2006).

In one embodiment, a plasmid including a Factor VIII-Fc fusion coding sequence and a selectable marker, e.g., zeocin resistance, is transfected into HEK 293 cells, for production of Factor VIII-Fc fusion.

In another embodiment, a first plasmid including a Factor VIII-Fc fusion coding sequence and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including an Fc coding sequence and a second selectable marker, e.g., a neomycin resistance gene, are cotransfected into HEK 293 cells, for production of Factor VIII-Fc monomer-dimer hybrid. The first and second plasmids can be introduced in equal amounts (i.e., 1:1 ratio), or they can be introduced in unequal amounts.

In some embodiments, a first plasmid including a Factor VIII-Fc fusion coding sequence and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including an Fc coding sequence and a second selectable marker, e.g., a neomycin resistance gene, and a third plasmid including a protein convertase coding sequence and a third selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of Factor VIII-Fc monomer-dimer hybrid. The first and second plasmids can be introduced in equal amounts (i.e., 1:1 molar ratio), or they can be introduced in unequal amounts.

In certain embodiments, a first plasmid, including a Factor VIII-Fc fusion coding sequence, an Fc coding sequence, and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including a protein convertase coding sequence and a second selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of Factor VIII-Fc monomer-dimer hybrid. The promoters for the Factor VIII-Fc fusion coding sequence and the Fc coding sequence can be different or they can be the same.

In other embodiments, a first plasmid, including a Factor VIII-Fc fusion coding sequence, an Fc coding sequence, and a first selectable marker, e.g., a zeocin resistance gene, and a second plasmid including an Fc coding sequence and a second selectable marker, e.g., a neomycin resistance gene, and a third plasmid including a protein convertase coding sequence and a third selectable marker, e.g., a hygromycin resistance gene, are cotransfected into HEK 293 cells, for production of Factor VIII-Fc monomer-dimer hybrid. The promoters for the Factor VIII-Fc fusion coding sequence and the Fc coding sequence in the first plasmid can be different or they can be the same. The first and second plasmids can be introduced in equal amounts (i.e., 1:1 molar ratio), or they can be introduced in unequal amounts.

In still other embodiments, transfected cells are stably transfected. These cells can be selected and maintained as a stable cell line, using conventional techniques known to those of skill in the art.

Host cells containing DNA constructs of the protein are grown in an appropriate growth medium. As used herein, the term “appropriate growth medium” means a medium containing nutrients required for the growth of cells. Nutrients required for cell growth may include a carbon source, a nitrogen source, essential amino acids, vitamins, minerals, and growth factors. Optionally the media can contain one or more selection factors. Optionally the media can contain bovine calf serum or fetal calf serum (FCS). In one embodiment, the media contains substantially no IgG. The growth medium will generally select for cells containing the DNA construct by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker on the DNA construct or co-transfected with the DNA construct. Cultured mammalian cells are generally grown in commercially available serum-containing or serum-free media (e.g., MEM, DMEM, DMEM/F12). In one embodiment the medium is CD293 (Invitrogen, Carlsbad, Calif.). In one embodiment, the medium is CD17 (Invitrogen, Carlsbad, Calif.). Selection of a medium appropriate for the particular cell line used is within the level of ordinary skill in the art.

In order to co-express Factor VIII and a proprotein convertase, the host cells are cultured under conditions that allow expression of both the Factor VIII and the functional proprotein convertase. As used herein, culturing refers to maintaining living cells in vitro for at least a definite time. Maintaining can, but need not include, an increase in population of living cells. For example, cells maintained in culture can be static in population, but still viable and capable of producing a desired product, e.g., a recombinant protein or recombinant fusion protein. Suitable conditions for culturing eukaryotic cells are well known in the art and include appropriate selection of culture media, media supplements, temperature, pH, oxygen saturation, and the like. For commercial purposes, culturing can include the use of any of various types of scale-up systems including shaker flasks, roller bottles, hollow fiber bioreactors, stirred-tank bioreactors, airlift bioreactors, Wave bioreactors, and others.

The cell culture conditions are also selected to allow processing of Factor VIII by the functional proprotein convertase. Conditions that allow processing of Factor VIII by the functional proprotein convertase specifically may include the presence of a source of vitamin K. For example, in one embodiment, stably transfected HEK 293 cells are cultured in CD293 media (Invitrogen, Carlsbad, Calif.) supplemented with 4 mM glutamine

In one aspect, the present invention is directed to a method of reducing nonprocessed Factor VIII in a culture medium expressing Factor VIII from a first polynucleotide in a host cell, where the endogenous processing enzymes of the host cell are insufficient to convert all of the Factor VIII to its processed isoform; b) expressing a proprotein convertase from a second polynucleotide in the host cell; and c) reducing the nonprocessed Factor VIII by processing with said proprotein convertase. In one embodiment, a present method reduces the nonprocessed Factor VIII completely, e.g., to an undetectable level as measured by SDS-PAGE. In another embodiment, a present method reduces the level of the nonprocessed Factor VIII partially or completely, e.g., less than 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% in the Factor VIII yield from the culture compared to the level of the nonprocessed Factor VIII without co-transfection with a proprotein convertase encoding gene, thereby generating a Factor VIII product comprising more than 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% processed Factor VIII.

The invention, in another aspect, relates to a method for increasing yield of a functional Factor VIII. As used herein, increasing yield refers to inducing a measurable increase in amount or activity of Factor VIII, obtained with a proprotein convertase and under specified conditions, as compared to a reference amount or activity of Factor VIII, obtained without a proprotein convertase and under the specified conditions. In one embodiment, the measurable increase is at least 5 percent. In another embodiment, the measurable increase is at least 10 percent. In some embodiments, the measurable increase is at least 20 percent, at least 30 percent, at least 40 percent, at least 50 percent, at least 60 percent, at least 70 percent, at least 80 percent, at least 90 percent, or at least 100 percent.

In further embodiments, the protein product containing the Factor VIII protein or a fusion protein thereof is secreted into the media. Media is separated from the cells, concentrated, filtered, and then passed over two or three affinity columns, e.g., a protein A column and one or two anion exchange columns.

In one aspect, the present invention relates to an isolated polypeptide comprising the Factor VIII protein or a fusion protein thereof produced by the methods described herein. For example, an isolated Factor VIII, which comprises expressing Factor VIII from a first polynucleotide in a host cell, wherein the endogenous processing enzymes of the host cell are insufficient to convert all of the Factor VIII to its processed isoform, expressing

Methods of Using Chimeric Proteins

The Factor VIII protein or a fusion protein thereof prepared by the invention has many uses as will be recognized by one skilled in the art, including, but not limited to methods of treating a subject having a hemostatic disorder and methods of treating a subject in need of a general hemostatic agent. In one embodiment, the invention relates to a method of treating a subject having a hemostatic disorder comprising administering a therapeutically effective amount of Factor VIII produced by the present invention.

The Factor VIII protein or a fusion protein thereof prepared by the invention treats or prevents a hemostatic disorder by serving as a cofactor to Factor IX on a negatively charged phospholipid surface, thereby forming a Xase complex. The binding of activated coagulation factors to a phospholipid surface localizes this process to sites of vascular damage. On a phospholipid surface, Factor VIIIa increases the maximum velocity of Factor X activation by Factor IXa, by approximately 200,000-fold, leading to the large second burst of thrombin generation.

The Factor VIII protein or a fusion protein thereof prepared by the invention can be used to treat any hemostatic disorder. The hemostatic disorders that may be treated by administration of Factor VIII of the invention include, but are not limited to, hemophilia A, as well as deficiencies or structural abnormalities relating to Factor VIII. In one embodiment, the hemostatic disorder is hemophilia A.

The Factor VIII protein or a fusion protein thereof prepared by the invention can be used to prophylactically treat a subject with a hemostatic disorder. The chimeric protein of the invention can be used to treat an acute bleeding episode in a subject with a hemostatic disorder. In another embodiment, the hemostatic disorder can be the result of a defective clotting factor, e.g., von Willebrand's factor. In one embodiment, the hemostatic disorder is an inherited disorder. In another embodiment, the hemostatic disorder is an acquired disorder. The acquired disorder can result from an underlying secondary disease or condition. The unrelated condition can be, as an example, but not as a limitation, cancer, an auto-immmune disease, or pregnancy. The acquired disorder can result from old age or from medication to treat an underlying secondary disorder (e.g. cancer chemotherapy).

The invention also relates to methods of treating a subject that does not have a hemostatic disorder or a secondary disease or condition resulting in acquisition of a hemostatic disorder. The invention thus relates to a method of treating a subject in need of a general hemostatic agent comprising administering a therapeutically effective amount of the Factor VIII protein or a fusion protein thereof prepared by the present methods.

In one embodiment, the subject in need of a general hemostatic agent is undergoing, or is about to undergo, surgery. The Factor VIII or a fusion protein thereof can be administered prior to, during, or after surgery as a prophylactic. The Factor VIII or a fusion protein thereof can be administered prior to, during, or after surgery to control an acute bleeding episode. The surgery can include, but is not limited to liver transplantation, liver resection, or stem cell transplantation.

The Factor VIII protein or a fusion protein thereof prepared by the invention can be used to treat a subject having an acute bleeding episode who does not have a hemostatic disorder. The acute bleeding episode can result from severe trauma, e.g., surgery, an automobile accident, wound, laceration gun shot, or any other traumatic event resulting in uncontrolled bleeding.

In one embodiment, the Factor VIII protein or a fusion protein thereof of the invention is administered intravenously, subcutaneously, intramuscularly, or via any mucosal surface, e.g., orally, sublingually, buccally, nasally, rectally, vaginally or via pulmonary route. The Factor VIII or a fusion protein thereof can be implanted within or linked to a biopolymer solid support that allows for the slow release of the chimeric protein to the site of bleeding or implanted into bandage/dressing. The dose of the Factor VIII protein or the fusion protein thereof will vary depending on the subject and upon the particular route of administration used. Dosages can range from 0.1 to 100,000 μg/kg body weight. In one embodiment, the dosing range is 0.1-1,000 μg/kg. The protein can be administered continuously or at specific timed intervals. In vitro assays may be employed to determine optimal dose ranges and/or schedules for administration. In vitro assays that measure clotting factor activity are known in the art, e.g., STA-CLOT Vila-rTF clotting assay. Additionally, effective doses may be extrapolated from dose-response curves obtained from animal models, e.g., a hemophiliac dog (Mount et al. 2002, Blood 99(8):2670).

The invention also relates to a pharmaceutical composition comprising Factor VIII or fusion protein of the invention. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E. W. Martin. Examples of excipients can include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. The composition can also contain pH buffering reagents, and wetting or emulsifying agents.

For oral administration, the pharmaceutical composition can take the form of tablets or capsules prepared by conventional means. The composition can also be prepared as a liquid for example a syrup or a suspension. The liquid can include suspending agents (e.g., sorbitol syrup, cellulose derivatives or hydrogenated edible fats), emulsifying agents (lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations can also include flavoring, coloring and sweetening agents. Alternatively, the composition can be presented as a dry product for constitution with water or another suitable vehicle.

For buccal administration, the composition may take the form of tablets or lozenges according to conventional protocols.

For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of a nebulized aerosol with or without excipients or in the form of an aerosol spray from a pressurized pack or nebulizer, with optionally a propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit can be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition can be formulated for parenteral administration (i.e. intravenous or intramuscular) by bolus injection. Formulations for injection can be presented in unit dosage form, e.g., in ampoules or in multidose containers with an added preservative. The compositions can take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient can be in powder form for constitution with a suitable vehicle, e.g., pyrogen free water.

The pharmaceutical composition can also be formulated for rectal administration as a suppository or retention enema, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In certain embodiments, the invention relates to a method of treating a subject with a hemostatic disorder comprising administering a therapeutically effective amount of Factor VIII or fusion proteins thereof prepared by the present invention in combination with at least one other clotting factor or-agent that promotes hemostasis. Said other clotting factor or agent that promotes hemostasis can be any therapeutic with demonstrated clotting activity. As an example, but not as a limitation, the clotting factor or hemostatic agent can include factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, prothrombin, fibrinogen, von Willebrand factor or recombinant soluble tissue factor (rsTF) or activated forms of any of the preceding. The clotting factor of hemostatic agent can also include anti-fibrinolytic drugs, e.g., epsilon-amino-caproic acid, tranexamic acid.

Kits

The invention provides a kit for the diagnosis of a hemostatic disorder. The kit can include a container and Factor VIII or fusion protein thereof. The Factor VIII or fusion proteins thereof can be provided in an appropriate buffer or solvent. The buffer can be an aqueous buffer, e.g., PBS or alternatively the chimeric protein can be lyophilized. The kit can also provide instructions for detecting the presence of a clotting factor in a sample, e.g., contacting an aliquot of a sample with the chimeric protein of the invention and detecting the presence of a clot. Detection can include visible detection. The kit can optionally provide an aliquot of blood lacking a known clotting factor.

Having now described the present invention in detail, the same will be more clearly understood by reference to the following examples, which are included herewith for purposes of illustration only and are not intended to be limiting of the invention. All patents and publications referred to herein are expressly incorporated by reference.

EXAMPLES Example 1. Cloning of Factor VIII-Fc Expressing Constructs

The coding sequence of human recombinant B-domain deleted FVIII was obtained by reverse transcription-polymerase chain reaction (RT-PCR) from human liver poly A RNA (Clontech) using FVIII-specific primers. The FVIII sequence includes the native signal sequence for FVIII. The B-domain deletion starts after serine 743 (5743; 2287 bp) and ends before glutamine 1638 (Q1638; 4969 bp) for a total deletion of 2682 bp (SQ version, which is represented by SEQ ID NO: 2).

The coding sequence for human recombinant Fc was obtained by RT-PCR from a human leukocyte cDNA library (Clontech) using Fc specific primers. Primers were designed such that the B-domain deleted FVIII sequence was fused directly to the N-terminus of the Fc sequence with no intervening linker. The FVIIIFc DNA sequence was cloned into the mammalian dual expression vector pBUDCE4.1 (Invitrogen) under control of the CMV promoter.

A second identical Fc sequence including the mouse Igk signal sequence was obtained by RT-PCR and cloned downstream of the second promoter, EF1α, in the expression vector pBUDCE4.1. This final construct was designated pSYN-FVIII-013.

A second plasmid was created from similar constructs using PCR and standard molecular biology techniques, in order to express rFVIIIBDD-Fc-Fc in which the rFVIIIBDDFc coding sequence was fused to the second Fc sequence with a (GGGGS)4 linker, allowing for production of only the rFVIIIBDD-Fc monomer-dimer hybrid in transient transfection. This construct was designated pSYN-FVIII-041.

Example 2: Cloning of PC5

The coding sequence for human PC5 was obtained by RT-PCR. The following primers were used (areas that anneal to the cDNA are indicated in bold):

SEQ Primers ID NO Sequences PC5-KpnI-F: 29 5′-ATCTACACCATCTCCA TCAGCAGC-3′ PC5 NotI-R: 30 5′-AAGGCGGCCGCTCAGC CTTGAAATGTACATGTTTT GC-3′ PC5-UTR-F: 31 5′-AGCGAGGGAGCAGCGA GG-3′ PC5-HindIII-R: 32 5′-GGTAGTTGACATGGCG GTTGG-3′ PC5-Af12-F: 33 5′-CAGCGACTTAAGCCAC CATGGGCTGGGGGAGCCG-3′ PC5-KpnI-R: 34 5′-GTAGGTTGTGGCCAGC GTGG-3′

Coding sequence for human PC5 (GenBank accession no. NM-006200) was obtained in two pieces. The 3′˜1750 bp were obtained using the primers PC5-KpnI-F and PC5-NotI-R with the Invitrogen SUPERSCRIPT™ RT-PCR with PLATINUM™ Taq kit according to the manufacturer's standard protocol, from human liver mRNA. The cycle used for the reverse transcription was 30 min at 50° C. followed by denaturing at 94° C. for 2 min and 35 cycles of 94° C. for 15 sec, 54° C. for 30 sec, 72° C. for 3 min, followed by 10 min extension at 72° C. and then storage at 4 ‘C. This produced a fragment from the internal KpnI site in the PC5 coding sequence through the stop codon, with a NotI site added at the 3’ end. This fragment was then cloned into pCR2.1 TOPO according to manufacturer's protocol to generate pSYN-PC5-001 (pCR2.1/PC5 (KpnI-NotI)). This fragment was then subcloned into pcDNA3.1/hygro using the KpnI and NotI restriction sites to generate pSYN-PC5-002 (pcDNA3.1/hygro/PC5 (KpnI-NotI)).

The 5′˜1100 bp of PC5 was obtained in two steps. It was first amplified by RT-PCR using the primers PC5-UTR-F and PC5-HindIII-R to amplify a ˜4520 bp fragment from human liver mRNA, using similar conditions as above, with an annealing temperature of 57° C. These primers have complete homology to the native PC5 sequence, in the untranslated 5′ sequence and sequence 3′ from the internal unique HindIII site, respectively. Note that this HindIII site is not present in the final construct due to a silent nucleotide substitution. This DNA fragment was then gel purified and used as a template for a second PCR reaction with PC5-Afl2-F, which adds an AflII cloning site followed by a Kozak sequence to the N-terminal coding sequence at the 5′ end, and PC5-KpnI-R, which anneals 3′ to the internal unique KpnI site, to generate an ˜1100 bp fragment. The reaction was carried out with the EXPAND™ High Fidelity System according to the manufacturer's standard protocol in a MJ Thermocycler using the following cycles: 94° C., 2 min; 14 cycles of (94° C., 30 sec, 57° C., 30 sec, 72° C., 2 min), followed by 72° C., 10 min. This fragment was then subcloned into pSYN-PC5-002 using the AflII and KpnI restriction sites to generate pSYN-PC5-003 (pcDNA3.1/hygro/PC5).

The nucleotide sequence encoding PC5 in pSYN-PC5-003 has the following sequence (SEQ ID NO: 35):

atgggctggg ggagccgctg ctgctgcccg ggacgtttgg acctgctgtg cgtgctggcg ctgctcgggg gctgcctgct ccccgtgtgt cggacgcgcg tctacaccaa ccactgggca gtcaaaatcg ccgggggctt cccggaggcc aaccgtatcg ccagcaagta cggattcatc aacataggac agataggggc cctgaaggac tactaccact tctaccatag caggacgatt aaaaggtcag ttatctcgag cagagggacc cacagtttca tttcaatgga accaaaggtg gaatggatcc aacagcaagt ggtaaaaaag cggacaaaga gggattatga cttcagtcgt gcccagtcta cctatttcaa tgatcccaag tggcccagta tgtggtatat gcactgcagt gacaatacac atccctgcca gtctgacatg aatatcgaag gagcctggaa gagaggctac acgggaaaga acattgtggt cactatcctg gatgacggaa ttgagagaac ccatccagat ctgatgcaaa actacgatgc tctggcaagt tgcgacgtga atgggaatga cttggaccca atgcctcgtt atgatgcaag caacgagaac aagcatggga ctcgctgtgc tggagaagtg gcagccgctg caaacaattc gcactgcaca gtcggaattg ctttcaacgc caagatcgga ggagtgcgaa tgctggacgg agatgtcacg gacatggttg aagcaaaatc agttagcttc aaccaccagc acgtgcacat ttacagcgcc agctggggcc cggatgatga tggcaagact gtggacggac cagcccccct cacccggcaa gcctttgaaa acggcgttag aatggggcgg agaggcctcg gctctgtgtt tgtttgggca tctggaaatg gtggaaggag caaagaccac tgctcctgtg atggctacac caacagcatc tacaccatct ccatcagcag cactgcagaa agcggaaaga aaccttggta cctggaagag tgttcatcca cgctggccac aacctacagc agcggggagt cctacgataa gaaaatcatc actacagatc tgaggcagcg ttgcacggac aaccacactg ggacgtcagc ctgagccccc atggctgcag gcatcattgc gctggccctg gaagccaatc cgtttctgac ctggagagac gtacagcatg ttattgtcag gacttcccgt gcgggacatt tgaacgctaa tgactggaaa accaatgctg ctggttttaa ggtgagccat ctttatggat ttggactgat ggacgcagaa gccatggtga tggaggcaga gaagtggacc accgttcccc ggcagcacgt gtgtgtggag agcacagacc gacaaatcaa gacaatccgc cctaacagtg cagtgcgctc catctacaaa gcctcaggct gctcagataa ccccaaccgc catgtcaact acctggagca cgtcgttgtg cgcatcacca tcacccaccc caggagagga gacctggcca tctacctgac ctcgccctct ggaactaggt ctcagctttt ggccaacagg ctatttgatc actccatgga aggattcaaa aactgggagt tcatgaccat tcattgctgg ggagaaagag ctgctggtga ctgggtcctt gaagtttatg atactccctc tcagctaagg aactttaaga ctccaggtaa attgaaagaa tggtctttgg tcctctacgg cacctccgtg cagccatatt caccaaccaa tgaatttccg aaagtggaac ggttccgcta tagccgagtt gaagacccca cagacgacta tggcacagag gattatgcag gtccctgcga ccctgagtgc agtgaggttg gctgtgacgg gccaggacca gaccactgca atgactgttt gcactactac tacaagctga aaaacaatac caggatctgt gtctccagct gcccccctgg ccactaccac gccgacaaga agcgctgcag gaagtgtgcc cccaactgtg agtcctgctt tgggagccat ggtgaccaat gcatgtcctg caaatatgga tactttctga atgaagaaac caacagctgt gttactcact gccctgatgg gtcatatcag gataccaaga aaaatctttg ccggaaatgc agtgaaaact gcaagacatg tactgaattc cataactgta gagaatgtag ggatgggtta agcctgcagg gatcccggtg ctctgtctcc tgtgaagatg gacggtattt caacggccag gactgccagc cctgccaccg cttctgcgcc acttgtgctg gggcaggagc tgatgggtgc attaactgca cagagggcta cttcatggag gatgggagat gcgtgcagag ctgtagtatc agctattact ttgaccactc ttcagagaat ggatacaaat cctgcaaaaa atgtgatatc agttgtttga cgtgcaatgg cccaggattc aagaactgta caagctgccc tagtgggtat ctcttagact taggaatgtg tcaaatggga gccatttgca aggatgcaac ggaagagtcc tgggcggaag gaggcttctg tatgcttgtg aaaaagaaca atctgtgcca acggaaggtt cttcaacaac tttgctgcaa aacatgtaca tttcaaggc

SEQ ID NO:35 contains substitutions from the GenBank sequence that do not affect the amino acid coding sequence. Specifically, the nucleotide at position 399 (corresponding to position 876 of GenBank accession no. NM-006200) is a T instead of a C, but preserves the amino acid Ser 133 (corresponding to amino acid numbering in GenBank accession no. NP-006191); nucleotide position 1473 (GenBank position 1950) is a C instead of a T, but preserves the amino acid Ala 491; and nucleotide position 1485 (GenBank position 1962) is an A instead of a G, but preserves the amino acid Ser 496. The nucleotide change at position 1473 eliminates a HindIII restriction site.

Example 3: Cloning of PACE-SOL

The coding sequence for human PACE was obtained by RT-PCR. The following primers were used (areas that anneal to the cDNA are indicated in bold):

SEQ ID NO PACE-F1: 36 5′-GGTAAGCTTGCCATGGAGCTG AGGCCCTGGTTGC-3′ PACE-R1: 37 5′-GTTTTCAATCTCTAGGACCCA CTCGCC-3′ PACE-F2: 38 5′-GCCAGGCCACATGACTACTCC GC-3′ PACE-R2: 39 5′-GGTGAATTCTCACTCAGGCAG GTGTGAGGGCAGC-3′

The primer PACE-F1 adds a HindIII site to the 5′ end of the PACE sequence beginning with 3 nucleotides before the start codon, while the primer PACE-R2 adds a stop codon after amino acid 715, which occurs at the end of the extracellular domain of PACE, as well as adding an EcoRI site to the 3′ end of the stop codon. The PACE-R1 and PACE-F2 primers anneal on the 3′ and 5′ sides of an internal BamHI site, respectively. Two RT-PCR reactions were then set up using 25 pmol each of the primer pairs of PACE-F1/R1 or PACE-F2/R2 with 20 ng of adult human liver RNA (Clontech; Palo Alto, Calif.) in a 50 μl RT-PCR reaction using the SUPERSCRIPT™ One-Step RT-PCR with PLATINUM® Taq system (Invitrogen, Carlsbad, Calif.) according to manufacturer's protocol. The reaction was carried out in a MJ Thermocycler using the following cycles: 50° C. 30 minutes; 94° C. 2 minutes; 30 cycles of (94° C. 30 seconds, 58° C. 30 seconds, 72° C. 2 minutes), followed by 72° C. 10 minutes. Each of these fragments was ligated into the vector pGEM T-Easy (Promega, Madison, Wis.) and sequenced fully. The F2-R2 fragment was then subcloned into pcDNA6 V5/His (Invitrogen, Carlsbad, Calif.) using the BamHI/EcoRI sites, and then the F1-R1 fragment was cloned into this construct using the HindIII/BamHI sites. The final plasmid, pcDNA6-PACE, produces a soluble form of PACE (amino acids 1-715), as the transmembrane region has been deleted. The sequence of PACE in pcDNA6-PACE is essentially as described in Harrison S et al., (1998) Semin Hematol 35(2 Suppl 2):4-10.

Example 4: Cloning of Kex2-SOL

The coding sequence for the yeast endoprotease, KEX2, was obtained by RT-PCR from Saccharomyces cerevisiae polyA+ mRNA (BD Clontech, cat #6999-1) using the following primers (areas that anneal to the cDNA are indicated in bold):

SEQ Primers ID NO Sequences KEX2-F: 40 5′-GCGCTAGCCGTACGGCCGCCACCA TGAAAGTGAGGAAATATATTACTTTAT GC-3′ KEX2-BglII-F: 41 5′-GCTATTGATCACAAAGATCTACAT CCTCC-3′ KEX2-BglII-R: 42 5′-GGAGGATGTAGATCTTTGTGATCA ATAGC-3′ KEX2-675-R: 43 5′-GCGAATTCCGGTCCGTCATTGCCT AGGGCTCGAGAGTTTTTTAGGAGTGTT TGGATCAG-3′

These primers were used to obtain coding sequence for KEX2 (amino acids 1-675), the yeast homolog to PACE, in two pieces in a manner similar to that used for PACE-SOL, Example 3 above; similarly, the transmembrane region was removed to generate the soluble form of the protein.

Example 5: Cloning of PC7-SOL

The coding sequence for PC7 was obtained by RT-PCR from human adult liver mRNA using the following primers (areas that anneal to the cDNA are indicated in bold):

SEQ ID NO PC7-BamMut-F: 44 5′-GCATGGACTCCGATCCCAACG-3′ PC7-BamMut-R: 45 5′-CGTTGGGATCGGAGTCCATGC-3′ PC7-F: 46 5′-GGTAAGCTTGCCGCCACCATGCCG AAGGGGAGGCAGAAAG-3′ PC7-SOL-R: 47 5′-TTTGAATTCTCAGTTGGGGGTGAT GGTGTAACC-3′ PC7-Xma-F: 48 5′-GGCACCTGAATAACCGACGG-3′ PC7-Xma-R: 49 5′-CGTCACGTTGATGTCCCTGC-3′

These primers were used to obtain coding sequence for PC7 (amino acids 1-663) in three pieces in a manner similar to that used for PACE-SOL, Example 3 above; similarly, the transmembrane region was removed to generate the soluble form of the protein.

Example 6: Transient Transfection of FVIIIFc Constructs in HEK293 Cells

HEK293 cells were transiently transfected with 25 μg pSYN-FVIII-041, or cotransfected with 12.5 μg pSYN-FVIII-041 and with 12.5 μg pSYN-PC5-003 or PACE expressing construct described in Example 3, yeast Kex2 expressing construct described in Example 5, or PC7 expressing construct described in Example 5 using PEI transfection reagent.

Example 7: Protein a Immunoprecipitation for SDS-PAGE and Western Blot Analysis

In order to analyze the FVIII-Fc from the transient transfection, the transfectants conditioned media on day 5 of transfection was subject to protein A immunoprecipitation. Specifically, conditioned media from HEK293 transfectants ((1) FVIIIFc alone, (2) FVIIIFc+Kex2, (3) FVIIIFc+PC7, (4) FVIIIFc+PACE, and (5) FVIIIFc+PC5) were supplemented with 1/10 volume of 5 ml of 4M NaCl, 5 mM CaCl2, and 10 mM HEPES buffer and incubated for 30 minutes. The cells were then centrifuged at 1000 rpm for 5 minutes. The collected supernatant was mixed with 25 μl of Protein A resin (Peirce #20338) and 50 ml conditioned media and incubated at 4° C. with rocking. The mixture was centrifuged for 5 minutes at 2,000 rpm, and the pellets were resuspended in 1 ml DPBS without Ca2+ and Mg2+. The resuspended mixture was again centrifuged for 30 minutes at 2,000 rpm, and the pellets were resuspended in 40 μl 2×TG SDS Sample loading buffer with a reducing agent. The resuspended mixture was heated for 5 minutes at 100° C.

Example 8: For Western Blot Analysis

For the Western blot analysis, 5 μl of the eluted mixture was loaded per lane on a 4-20% SDS TG gel. The gel was transferred to Nitrocellulose membrane (whatman Protran cat #10401396) at 25 V constant for 1.5 hours. For detection of the heavy chain alone, the membrane was incubated in 15 ml blocking buffer with 15 μl mouse monoclonal a-human FVIII A2 Domain antibody at 1:1000 dilution (cat # GMA-012) at 4° C., washed 3×5 min in PBST, followed by incubation with goat anti-mouse IgG peroxidase conjugate at 1:10,000 in 15 ml blocking buffer for 30 min, washed 3×5 min with PBST, and then incubated with then in ECL plus Western blotting detection system (GE Healthcare, cat # RPN2132) for five minutes. The membrane was then scanned.

For detection of the light chain (LC-Fc), anti-human IgG (Fc specific)-horseradish peroxidase conjugate was used at 1:10,000 dilution (Thermo Scientific cat #31413) at 4° C. The membrane was then washed 3×5 minutes in PBST at room temperature and then incubated in ECL plus Western blotting detection system (GE Healthcare, cat # RPN 2132) for five minutes.

As FIGS. 3A and 3B show, the HEK293 cells transfected only with the FVIIIFc expressing construct shows nonprocessed FVIIIFc (NP) as well as processed FVIIIFc (LC-Fc and HC) while the HEK293 cells co-transfected with the FVIIIFc expressing construct and the Kex2 expressing construct (lane 2), the PC7 expressing constructs (lane 3), the PACE expressing construct (lane 4), or the PC5 expressing construct (lane 5) show no nonprocessed Factor VIII, only LC-Fc and HC. These figures also indicated that the HEK293 cells co-transfected with the FVIIIFc expressing construct and the Kex2 expressing construct (lane 2) or the PACE expressing construct (lane 4) result in lower levels of the FVIII heavy chain, possibly due to degradation by the processing enzyme, while the HEK293 cells co-transfected with the PC7 expressing constructs (lane 3) or the PC5 expressing construct (lane 5) show relatively higher recovery of the heavy chain. Note that the lane with FVIIIFc alone was generated with twice the amount of FVIIIFc construct DNA, therefore a direct comparison to the amount of reduction of yield by PC7 and PC5 is difficult to make. In all cases the processing enzyme eliminated the amount of nonprocessed FVIII.

Example 10: SYPRO™ Ruby Gel Staining and Coomassie Gel Staining

Alternative to the Western blotting, an SDS-PAGE gel was run with 6.7 μl of eluted material and was stained with SYPRO RUBY™ (Bio-Rad Laboratories), imaged, and then stained with Coomassie blue. As FIGS. 4A and 4B show, SYPRO RUBY™ gel staining and Coomassie gel staining show consistent results that Kex2, PC7, PACE, and PC5 decrease nonprocessed Factor VIII. Similarly, the staining methods also confirm lower levels of the FVIII heavy chain by cotransfection with Kex2 and PACE, but relatively higher levels of FVIII heavy chain by cotransfection with PC7 and PC5.

Example 11: Stable Transfection in HEK293 Cells

A stable cell line expressing rFVIIIFc (3C4-22) was generated by transfecting HEK293 cells with pSYN-FVIII-013 described in Example 1 using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, Calif.) according to manufacturers protocols. Stable cell lines were selected with 200 μg/mL zeocin, adapted to serum free suspension; line 3C4 was chosen for cloning due to high levels of expression, and line 3C4-22 was ultimately chosen due to high levels of expression, growth profile, and stability.

Stable Factor VIII-Fc expressing cell line 3C4-22 was further transfected with pSYN-PC5-003 After 48 hours, cells were distributed in 96 well plates (2500-10000 cells/well), and stable lines selected with 50 μg/ml hygromycin. Cell lines were adapted to serum free suspension culture, and the cell line with best growth properties and expression levels (1D9) was chosen for further analysis.

Cell lines 3C4-22 and 3C4-22/PC5-003 1D9 were both expanded and rFVIIIFc protein purified from the cell culture media using a combination of affinity chromatography and standard chromatographic techniques.

Example 12: SDS PAGE Analysis of rFVIIIFc and rFVIIIFc/PC5

Protein was analyzed by both reducing and nonreducing SDS-PAGE. Lane 1 was contains rFVIIIFc purified from 3C4-22/PC5-003 1D9, lane 2 contains rFVIIIFc purified from 3C4-22. FIGS. 5A and 5B show that the FVIIIFc produced from line 3C4-22 (HEK293 cells transfected only with the FVIIIFc expressing construct) shows nonprocessed FVIIIBDDFc (NP) as well as processed FVIIIBDDFc (LC-Fc/LC-Fc2 and HC), but the FVIIIFc produced from line 3C4-22/PC5-003 1D9 (HEK293 cells co-transfected with the FVIIIFc expressing construct and PC5 expressing construct) express

Example 13. Cloning of BDD Factor VIII Expressing Construct

The coding sequence of human recombinant B-domain deleted FVIII was obtained by PCR from pSYN-FVIII-041 using specific primers designed to express the coding sequence for the BDD FVIII alone, without Fc, and cloned into the mammalian expression vector pcDNA4, under control of the CMV promoter.

Example 14: Transient Transfection of FVIII Construct in HEK293 Cells, and FVIII-Affinity Resin Pull Down

HEK293 cells were transiently transfected with 37.5 μg pSYN-FVIII-066, or cotransfected with 18.75 μg pSYN-FVIII-066 and with 18.75 μg pSYN-PC5-003 or PACE expressing construct described in Example 3, yeast Kex2 expressing construct described in Example 5, or PC7 expressing construct described in Example 5 using a modified protocol with FREESTYLE™ MAX reagent (Invitrogen, Carlsbad Calif.). Briefly, FreeStyle 293-F cells were split to 6-7×10⁵ cells/ml 24 hours before transfection.

Plasmid DNAs (37.5 μg) were diluted into OPTIPRO™ SFM to a total volume of 0.6 ml and further mixed with diluted FREESTYLE™ MAX Reagent to obtain a total volume of 1.2 ml. The DNA-lipid mixture were incubated for 10-20 minutes at room temperature to allow complexes to form and then added into each of 125 ml flasks containing 30 ml of the FREESTYLE™ 293-F cells prepared as shown above. The cells were incubated at 37° C., 8% CO2 on an orbital shaker set to 135 rpm.

Example 15: FVIII Affinity Resin Pull Down for SDS-PAGE and Western Blot Analysis

In order to analyze the FVIII from the transient transfection, the transfectants conditioned media on day 5 of transfection was subject to small scale FVIII Affinity resin pull down. Specifically, conditioned media from HEK293 transfectants ((1) FVIII alone, (2) FVIII+Kex2, (3) FVIII+PC7, (4) FVIII+PACE, and (5) FVIII+PC5) were supplemented with 1/10 volume of 3 ml of 4M NaCl, 5 mM CaCl2, and 10 mM HEPES buffer and incubated for 30 minutes. The cells were then centrifuged at 1000 rpm for 5 minutes. The FVIII protein was then purified by adding 15 μl of FVIIISelect Resin (GE Healthcare #17-5450-01) to 14 ml conditioned media and incubated at 4° C. with rotation overnight. The mixture was centrifuged for 5 minutes at 2,000 rpm, and the pellets were resuspended in 1 ml DPBS without Ca2+ and Mg2+. The resuspended mixture was transferred to eppendorf tubes, washed three times with 1 ml DBS, split into two tubes, and the final pellets resuspended in 23 μl 1×TG SDS Sample loading buffer with a reducing agent. The resuspended mixture was heated for 9 minutes at 100° C.

Example 16: For Western Blot Analysis

For the Western blot analysis, the 23 μl from the split tubes were loaded in each lane of a 4-20% SDS TG gel, and two gels analyzed in parallel. The gels were transferred to Nitrocellulose membrane using the iBlot (Invitrogen) standard protocol. For detection of the heavy chain alone, the membrane was incubated in 15 ml blocking buffer with 15 μl mouse monoclonal a-human FVIII A2 Domain antibody at 1:1000 dilution (cat # GMA-012) at 4° C., washed 3×5 min in PBST, followed by incubation with goat anti-mouse IgG peroxidase conjugate at 1:10,000 in 15 ml blocking buffer for 30 min, washed 3×5 min with PBST, and then incubated with ECL plus Western blotting detection system (GE Healthcare, cat # RPN2132) for five minutes. For the detection of the light chain, a similar analysis was performed, using a monoclonal anti-human FVIII antibody against the N-terminal region of the 83 kD light chain of Factor VIII (cat #10101-10104, QEDBioscience Inc) at 1:10,000 dilution. The membranes were then scanned. As FIGS. 6A and 6B show, the HEK293 cells transfected only with the FVIII expressing construct shows nonprocessed FVIII (NP) as well as processed FVIII (LC and HC) while the HEK293 cells co-transfected with the FVIII expressing construct and the Kex2 expressing construct (lane 2), the PACE expressing construct (lane 4), or the PC5 expressing construct (lane 5) show no nonprocessed Factor VIII, and the PC7 cotransfection (lane 3) showed reduced nonprocessed isoform. These figures also indicated that the HEK293 cells co-transfected with the FVIII expressing construct and the Kex2 expressing construct (lane 2) resulted in almost undetectable levels of the HC, and the PACE expressing construct (lane 4) resulted in lower levels of the FVIII heavy chain, possibly due to degradation by the processing enzyme, compared to HEK293 cells co-transfected with the PC7 expressing constructs (lane 3) or the PC5 expressing construct (lane 5), which show higher recovery of the heavy chain by comparison. Note that these pulldowns were performed with FVIIISelect affinity resin, which binds to FVIII, and therefore it cannot conclude whether the Kex2 cotransfection lead to greater degradation of the HC, or cleaved it in such a way that the FVIII was no longer able to interact with the resin. Note that the lane with FVIII alone was generated with twice the amount of FVIII construct DNA, therefore a direct comparison to the amount of reduction of yield by PC7 and PC5 is difficult to make. In all cases the processing enzyme reduced or eliminated the amount of nonprocessed FVIII.

Tables

TABLE 1 Polynucleotide Sequences A. B-Domain Deleted FVIIIFc (i) B-Domain Deleted FVIIIFc Chain DNA Sequence (FVIII signal peptide  underlined, Fc region in bold) (SEO ID NO: 59, which encodes SEQ ID NO: 60)  661                                A TGCAAATAGA GCTCTCCACC TGCTTCTTTC  721 TGTGCCTTTT GCGATTCTGC TTTAGTGCCA CCAGAAGATA CTACCTGGGT GCAGTGGAAC  781 TGTCATGGGA CTATATGCAA AGTGATCTCG GTGAGCTGCC TGTGGACGCA AGATTTCCTC  841 CTAGAGTGCC AAAATCTTTT CCATTCAACA CCTCAGTCGT GTACAAAAAG ACTCTGTTTG  901 TAGAATTCAC GGATCACCTT TTCAACATCG CTAAGCCAAG GCCACCCTGG ATGGGTCTGC  961 TAGGTCCTAC CATCCAGGCT GAGGTTTATG ATACAGTGGT CATTACACTT AAGAACATGG 1021 CTTCCCATCC TGTCAGTCTT CATGCTGTTG GTGTATCCTA CTGGAAAGCT TCTGAGGGAG 1081 CTGAATATGA TGATCAGACC AGTCAAAGGG AGAAAGAAGA TGATAAAGTC TTCCCTGGTG 1141 GAAGCCATAC ATATGTCTGG CAGGTCCTGA AAGACAATGG TCCAATGGCC TCTGACCCAC 1201 TGTGCCTTAC CTACTCATAT CTTTCTCATG TGGACCTGGT AAAAGACTTG AATTCAGGCC 1261 TCATTGGAGC CCTACTAGTA TGTAGAGAAG GGAGTCTGGC CAAGGAAAAG ACACAGACCT 1321 TGCACAAATT TATACTACTT TTTGCTGTAT TTGATGAAGG GAAAAGTTGG CACTCAGAAA 1381 CAAAGAACTC CTTGATGCAG GATAGGGATG CTGCATCTGC TCGGGCCTGG CCTAAAATGC 1441 ACACAGTCAA TGGTTATGTA AACAGGTCTC TGCCAGGTCT GATTGGATGC CACAGGAAAT 1501 CAGTCTATTG GCATGTGATT GGAATGGGCA CCACTCCTGA AGTGCACTCA ATATTCCTCG 1561 AAGGTCACAC ATTTCTTGTG AGGAACCATC GCCAGGCGTC CTTGGAAATC TCGCCAATAA 1621 CTTTCCTTAC TGCTCAAACA CTCTTGATGG ACCTTGGACA GTTTCTACTG TTTTGTCATA 1681 TCTCTTCCCA CCAACATGAT GGCATGGAAG CTTATGTCAA AGTAGACAGC TGTCCAGAGG 1741 AACCCCAACT ACGAATGAAA AATAATGAAG AAGCGGAASA CTATGATGAT GATCTTACTC 1801 ATTCTGAAAT GGATGTGGTC AGGTTTCATG ATGACAACTC TCCTTCCTTT ATCCAAATTC 1861 GCTCAGTTGC CAAGAAGCAT CCTAAAACTT GGGTACATTA CATTGCTGCT GAAGAGGAGG 1921 ACTGGGACTA TGCTCCCTTA GTCCTCGCCC CCGATGACAG AAGTTATAAA AGTCAATATT 1981 TGAACAATGG CCCTCAGCGG ATTGGTAGGA AGTACAAAAA AGTCCGATTT ATGGCATACA 2041 CAGATGAAAC CTTTAAGACT CGTGAAGCTA TTCAGCATGA ATCAGGAATC TTGGGACCTT 2101 TACTTTATGG GGAAGTTGGA GACACACTGT TGATTATATT TAAGAATCAA GCAAGCAGAC 2161 CATATAACAT CTACCCTCAC GGAATCACTG ATGTCCGTCC TTTGTATTCA AGGAGATTAC 2221 CAAAAGGTGT AAAACATTTG AAGGATTTTC CAATTCTGCC AGGAGAAATA TTCAAATATA 2281 AATGGACAGT GACTGTAGAA GATGGGCCAA CTAAATCAGA TCCTCGGTGC CTGACCCGCT 2341 ATTACTCTAG TTTCGTTAAT ATGGAGAGAG ATCTAGCTTC AGGACTCATT GGCCCTCTCC 2401 TCATCTGCTA CAAAGRATCT GTAGATCAAA GAGGAAACCA GATAATGTCA GACAAGAGGA 2461 ATGTCATCCT GTTTTCTGTA TTTGATGAGA ACCGAAGCTG GTACCTCACA GAGAATATAC 2521 AACGCTTTCT CCCCAATCCA GCTGGAGTGC AGCTTGAGGA TCCAGAGTTC CAAGCCTCCA 2581 ACATCATGCA CAGCATCAAT GGCTATGTTT TTGATAGTTT GCAGTTGTCA GTTTGTTTGC 2641 ATGAGGTGGC ATACTGGTAC ATTCTAAGCA TTGGAGCACA GACTGACTTC CTTTCTGTCT 2701 TCTTCTCTGG ATATACCTTC AAACACAAAA TGGTCTATGA AGACACACTC ACCCTATTCC 2761 CATTCTCAGG AGAAACTGTC TTCATCTCCA TGGAAAACCC AGGTCTATGG ATTCTGGGGT 2821 GCCACAACTC AGACTTTCGG AACAGAGGCA TGACCGCCTT ACTGAAGGTT TCTAGTTGTG 2881 ACAAGRACAC TGGTGATTAT TACGAGGACA GTTATGAAGA TATTTCAGCA TACTTGCTGA 2941 GTAAAAACAA TGCCATTGAA CCAAGAAGCT TCTCTCAAAA CCCACCAGTC TTGAAACGCC 3001 ATCAACGGGA AATAACTCCT ACTACTCTTC AGTCAGATCA AGAGGAAATT GACTATGATG 3061 ATACCATATC AGTTGAAATG AAGAAGGAAG ATTTTGACAT TTATGATGAG GATGAAAATC 3121 AGAGCCCCCG CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC 3181 TCTGGGATTA TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA 3241 GTGTCCCTCA GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATGGCTCC TTTACTCAGC 3301 CCTTATACCG TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG 3361 AAGTTGAAGA TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT 3421 ATTCTAGCCT TATTTCTTAT GAGGAAGATC AGAGGCAACC ACCAGAACCT AGAAAAAACT 3481 TTGTCAAGCC TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA 3541 CTAAAGATGA GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG 3601 ATGTGCACTC AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG 3661 CTCATGGGAG ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA 3721 CCAAAAGCTG GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC 3781 AGATGGAAGA TCCCACTTTT AAAGAGAATT ATCCCTTCCA TGCAATCAAT GGCTACATAA 3841 TGGATACACT ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA 3901 GCATGGGCAG CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC 3961 GAAAAAAAGA GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG 4021 TGGAAATGTT ACCATCCAAA GCTGGAATTT GGCGGGTGSA ATGCCTTATT GGCGAGCATC 4081 TACATGCTGG GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG 4141 GAATGGCTTC TGGACACATT AGAGATTTTC AGATTACAGC TTCAGGACAA TATGGACAGT 4201 GGGCCCCAAA CCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTGG RGCACCAAGG 4261 AGCCCTTTTC TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC GGCATCAAGA 4321 CCCAGGGTGC CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA 4381 GTCTTGATGG GAAGAAGTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT 4441 TCTTTGGCAA TGTCGATTCA TCTGGGATAA AACACAATAT TTTTAACCCT CCAATTATTG 4501 CTCGATACAT CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT 4561 TGATGGGCTG TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGAGT AAAGCAATAT 4621 CAGATGCACA GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT 4681 CAAAAGCTCG ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC 4741 CAAAAGAGTG GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC 4801 AGGGAGTAAA ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC 4861 AAGATGGCCA TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA 4921 ATCAAGACTC CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC 4981 TTCGAATTCA CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT 5041 GCGAGGCACA CSACCTCTAC GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCAGAAC 5101 TCCTGGGCGG ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT 5161 CCCGGACCCC TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA 5221 AGTTCAACTG GTACGTGGAC GGCGTGGAGG TGCAMAATGC CAAGACAAAG CCGCGGGAGG 5281 AGCAGTACAA CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC 5341 TGAATGGCAA GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA 5401 AAACCATCTC CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT 5461 CCCGGGATGA GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC 5521 CCAGCGACAT CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA 5581 CGCCTCCCGT GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA 5641 AGAGCAGGTG GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA 5701 ACCACTACAC GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A (ii) Fc DNA sequence (mouse Igκ signal peptide underlined)  (SEQ ID NO: 3, which encodes SEQ ID NO: 4) 7981                                                  ATGGA GACAGACACA 8041 CTCCTGCTAT GGGTACTGCT GCTCTGGGTT CCAGGTTCCA CTGGTGACAA AACTCACACA 8101 TGCCCACCGT GCCCAGCACC TGAACTCCTG GGAGGACCGT CAGTCTTCCT GTTCCCCCCA 8161 AAACCCAAGG ACACCCTCAT GATCTCCCGG ACCCCTGAGG TCACATGCGT GGTGGTGGAC 8221 GTGAGCCACG AAGACCCTGA GGTCAAGTTC AACTGGTACG TGGACGGCGT GGAGGTGCAT 8281 AATGCCAAGA CAAAGCCGCG GGAGGAGCAG TACAACAGCA CGTACCGTGT GGTCAGCGTC 8341 CTCACCGTCC TGCACCAGGA CTGGCTGAAT GGCAAGGAGT ACAAGTGCAA GGTCTCCAAC 8401 AAAGCCCTCC CAGCCCCCAT CGAGAAAACC ATCTCCAAAG CCAAAGGGCA GCCCCGAGAA 8461 CCACACCTGT ACACCCTGCC CCCATCCCGC GATGAGCTGA CCAAGAACCA GGTCAGCCTG 8521 ACCTGCCTGG TCAAAGGCTT CTATCCCAGC GACATCGCCG TGGAGTGGGA GAGCAATGGG 8581 CAGCCGGAGA ACAACTACAA GACCACGCCT CCCGTGTTGG ACTCCGACGG CTCCTTCTTC 8641 CTCTACAGCA AGCTCAGCGT GGACAAGAGC AGGTGGCAGC AGGGGAACGT CTTCTCATGC 8701 TCCGTGATGC ATGAGGCTCT GCACAACCAC TACACGCAGA AGAGCCTCTC CCTGTCTCCG 8761 GGTAAA B. Full-length FVIIIFc (i) Full-length FVIIIFc DNA Sequence (FVIII signal peptide underlined,  Fc region in bold) (SEQ ID NO: 61, which encodes SEQ ID NO: 62)  661                                         ATG CAAATAGAGC TCTCCACCTG  721 CTTCTTTCTG TGCCTTTTGC GATTCTGCTT TAGTGCCACC AGAAGATACT ACCTGGGTGC  781 AGTGGAACTG TCATGGGACT ATATGCAAAG TGATCTCGGT GAGCTGCCTG TGGACGCAAG  841 ATTTCCTCCT AGAGTGCCAA AATCTTTTCC ATTCAACACC TCACTCGTGT ACAAAAAGAC  901 TCTGTTTGTA GAATTCACGG ATCACCTTTT CAACATCGCT AAGCCAAGGC CACCCTGGAT  961 GGGTCTGCTA GGTCCTACCA TCCAGGCTGA GGTTTATGAT ACAGTGGTCA TTACACTTAA 1021 GAACATGGCT TCCCATCCTG TCAGTCTTCA TGCTGTTGGT GTATCCTACT GGAAAGCTTC 1081 TGAGGGAGCT GAATATGATC ATCAGACCAG TCAAAGGGAG AAAGAAGATG ATAAAGTCTT 1141 CCCTGGTGGA AGCCATACAT ATGTCTGGCA GGTCCTGAAA GAGAATGGTC CAATGGCCTC 1201 TGACCCACTG TGCCTTACCT ACTCATATCT TTCTCATGTG GACCTGGTAA AAGACTTGAA 1261 TTCAGGCCTC ATTGGAGCCC TACTAGTATG TAGAGAAGGG AGTCTGGCCA AGGAAAAGAC 1321 ACAGACCTTG CACAAATTTA TACTACTTTT TGCTGTATTT GATGAAGGGA AAAGTTGGCA 1381 CTCAGAAACA AAGAACTCCT TGATGCAGGA TAGGGATGCT GCATCTGCTC GGGCCTGGCC 1441 TAAAATGCAC ACAGTCAATG GTTATGTAAA CAGGTCTCTG CCAGGTCTGA TTGGATGCCA 1501 CAGGAAATCA GTCTATTGGC ATGTGATTGG AATGGGCACC ACTCCTGAAG TGCACTCAAT 1561 ATTCCTCGAA GGTCACACAT TTCTTGTGAG GAACCATCGC CAGGCGTCCT TCGAAATCTC 1621 GCCAATAACT TTCCTTACTG CTCAAACACT CTTGATGGAC CTTGaACAGT TTCTACTGTT 1681 TTGTCATATC TCTTCCCACC AACATGATGG CATGGAAGCT TATGTCAAAG TAGACAGCTG 1741 TCCAGAGGAA CCCCAACTAC GAATGAAAAA TAATGAAGAA GCGGAAGACT ATGATGATCA 1801 TCTTACTGAT TCTGAAATGC ATCTGGTCAG GTTTGATGAT GACAACTCTC CTTCCTTTAT 1861 CCAAATTCGC TCAGTTGCCA AGAAGCATCC TAAAACTTGG GTACATTACA TTGCTGCTGA 1921 AGAGGAGGAC TGGGACTATG CTCCCTTAGT CCTCGCCCCC GATGACAGAA GTTATAAAAG 1981 TCAATATTTG AACAATGGCC CTCAGCGGAT TGGTAGGAAG TACAAAAAAG TCCGATTTAT 2041 GGCATACACA GATGAAACCT TTAAGACTCG TGAACCTATT CAGCATGAAT GAGGAATCTT 2101 GGGACCTTTA CTTTATGGGG AAGTTGGAGA CACACTGTTG ATTATATTTA ACAATCAAGC 2161 AAGCAGACCA TATAACATCT ACCCTCACGG AATCACTGAT GTCCGTCCTT TGTATTCAAG 2221 GAGATTACCA AAAGGTGTAA AACATTTGAA GGATTTTCCA ATTCTGCCAG GAGAAATATT 2281 CAAATATAAA TGGACAGTGA CTGTAGAAGA TGGGCCAACT AAATCAGATC CTCGGTGCCT 2341 GACCCGCTAT TACTCTAGTT TCGTTAATAT GGAGAGAGAT CTACCTTCAC CACTCATTGG 2401 CCCTCTCCTC ATCTGCTACA AAGAATCTGT AGATCAAAGA GGAAACCAGA TAATGTCAGA 2461 CAAGAGGAAT GTCATCCTGT TTTCTGTATT TGATGAGAAC CGAAGCTGGT ACCTCACAGA 2521 GAATATACAA CGCTTTCTCC CCAATCCAGC TGGAGTGCAG CTTGAGGATC CAGAGTTCCA 2581 AGCCTCCAAC ATCATGCACA GCATCAATGG CTATGTTTTT GATAGTTTGC AGTTGTCAGT 2641 TTGTTTGCAT GAGGTGGCAT ACTGGTACAT TCTAAGCATT GGAGCACAGA CTGACTTCCT 2701 TTCTGTCTTC TTCTCTGGAT ATACCTTCAA ACACAAAATG GTCTATGAAG ACACACTCAC 2761 CCTATTCCCA TTCTCAGGAG AAACTGTCTT CATGTCGATG GAAAACCCAG GTCTATGGAT 2821 TCTGGGGTGC CACAACTCAG ACTTTCGGAA CAGAGGCATG ACCGCCTTAC TGAAGGTTTC 2881 TAGTTGTGAC AAGAACACTG GTGATTATTA CGAGGACAGT TATGAAGATA TTTCAGCATA 2941 CTTGCTGAGT AAAAACAATG CCATTGAACC AAGAAGCTTC TCCCAGAATT CAAGACACCC 3001 TAGCACTAGG CAAAAGCAAT TTAATGCCAC CACAATTCCA GAAAATGACA TAGAGAAGAC 3061 TGACCCTTGG TTTGCACACA GAACACCTAT GCCTAAAATA CAAAATGTCT CCTCTAGTGA 3121 TTTGTTGATG CTCTTGCGAC AGAGTCCTAC TCCACATGGG CTATCCTTAT CTGATCTCCA 3181 AGAAGCCAAA TATGAGACTT TTTCTGATGA TCCATCACCT GGAGCAATAG ACAGTAATAA 3241 CAGCCTGTCT GAAATGACAC ACTTCAGGCC ACAGCTCCAT CACAGTGGGG ACATGGTATT 3301 TACCCCTGAG TCAGGCCTCC AATTAAGATT AAATGAGAAA CTCGCGACAA CTGCAGCAAC 3361 AGAGTTGAAG AAACTTGATT TCAAAGTTTC TAGTACATCA AATAATCTGA TTTCAACAAT 3421 TCCATCAGAC AATTTGGCAG CAGGTACTGA TAATACAAGT TCCTTAGGAC CCCCAAGTAT 3481 GCCAGTTCAT TATGATAGTC AATTAGATAC CACTCTATTT GGCAAAAAGT CATCTCCCCT 3541 TACTGAGTCT GGTGGACCTC TGAGCTTGAG TGAAGAAAAT AATGATTCAA AGTTGTTAGA 3601 ATCAGGTTTA ATGAATAGCC AAGAAAGTTC ATGGGGAAAA AATGTATCGT CAACAGAGAG 3661 TGGTAGGTTA TTTAAAGGGA AAAGAGCTCA TGGACCTGCT TTGTTGACTA AAGATAATGC 3721 CTTATTCAAA GTTAGCATCT CTTTGTTAAA GACAAACAAA ACTTCCAATA ATTCAGCAAC 3781 TAATAGAAAG ACTCACATTG ATCGCCCATC ATTATTAATT GAGAATAGTC CATCAGTCTG 3841 GCAAAATATA TTAGAAAGTG ACACTGAGTT TAAAAAAGTG ACACCTTTGA TTCATGACAG 3901 AATGCTTATG GACAAAAATG CTACAGCTTT GAGGCTAAAT CATATGTCAA ATAAAACTAC 3961 TTCATCAAAA AACATGGAAA TGGTCCAACA GAAAAAAGAG GGCCCCATTC CACCAGATGC 4021 ACAAAATCCA GATATGTCGT TCTTTAAGAT GCTATTCTTG CCAGAATCAG CAAGGTGGAT 4081 ACAAAGGACT CATGGAAACA ACTCTCTGAA CTCTGGGCAA GGCCCCAGTC CAAAGCAATT 4141 AGTATCCTTA GGACCAGAAA AATCTGTGGA AGGTCAGAAT TTCTTGTCTG AGAAAAACAA 4201 AGTGGTAGTA GGAAAGGGTG AATTTACAAA GGACGTAGGA CTCAAAGAGA TGGTTTTTCC 4261 AAGCAGCAGA AACCTATTTC TTACTAACTT GGATAATTTA CATGAAAATA ATACACACAA 4321 TCAAGAAAAA AAAATTCAGG AAGAAATAGA AAAGAAGGAA ACATTAATCC AAGAGAATGT 4381 AGTTTTGCCT CAGATACATA CAGTGACTGG CACTAAGAAT TTCATGAAGA ACCTTTTCTT 4441 ACTGAGCACT AGGCAAAATG TAGAAGGTTC ATATGACGGG GCATATGCTC CAGTACTTCA 4501 AGATTTTAGG TCATTAAATG ATTCAACAAA TAGAACAAAG AAACACACAG CTCATTTCTC 4561 AAAAAAAGGG GAGGAAGAAA ACTTGGAAGG CTTGGGAAAT CAAACCAAGC AAATTGTAGA 4621 GAAATATGCA TGCACCACAA GGATATCTCC TAATACAAGC CAGCAGAATT TTGTCACGCA 4681 ACGTAGTAAG AGAGCTTTGA AACAATTCAG ACTCCCACTA GAAGAAACAG AACTTGAAAA 4741 AAGGATAATT GTGGATGACA CCTCAACCCA GTGGTCCAAA AACATGAAAC ATTTGACCCC 4801 GAGCACCCTC ACACAGATAG ACTACAATGA GAAGGAGAAA GGGGCCATTA CTCAGTCTCC 4861 CTTATCAGAT TGCCTTACGA GGAGTCATAG CATCCCTCAA GCAAATAGAT CTCCATTACC 4921 CATTGCAAAG GTATCATCAT TTCCATCTAT TAGACCTATA TATCTGACCA GGGTCCTATT 4981 CCAAGACAAC TCTTCTCATC TTCCAGCAGC ATCTTATAGA AAGAAAGATT CTGGGGTCCA 5041 AGAAAGCAGT CATTTCTTAC AAGGAGCCAA AAAAAATAAC CTTTCTTTAG CCATTCTAAC 5101 CTTGGAGATG ACTGGTGATC AAAGAGAGGT TGGCTCCCTG GGGACAAGTG CCACAAATTC 5161 AGTCACATAC AAGAAAGTTG AGAACACTGT TCTCCCGAAA CCAGACTTGC CCAAAACATC 5221 TGGCAAAGTT GAATTGCTTC CAAAAGTTCA CATTTATCAG AAGGACCTAT TCCCTACGGA 5281 AACTAGCAAT GGGTCTCCTG GCCATCTGGA TCTCGTGGAA GGGAGCCTTC TTCAGGGAAC 5341 AGAGGaAGCG ATTAAGTGGA ATGAACCAAA CAGACCTGGA AAAGTTCCCT TTCTGAGAGT 5401 AGCAACAGAA AGCTCTGCAA AGACTCCCTC CAAGCTATTG GATCCTCTTG CTTGGGATAA 5461 CCACTATGGT ACTCAGATAC CAAAAGAAGA GTGGAAATCC CAAGAGAAGT CACCAGAAAA 5521 AACAGCTTTT AAGAAAAAGG ATACCATTTT GTCCCTGAAC GCTTGTGAAA GCAATCATGC 5581 AATAGCAGCA ATAAATGAGG GACAAAATAA GCCCGAAATA GAAGTCACCT GGGCAAAGCA 5641 AGGTAGGACT GAAAGGCTGT GCTCTCAAAA CCCACCAGTC TTGAAACGCC ATCAACGGGA 5701 AATAACTCGT ACTACTCTTC AGTCAGATCA AGACCAAATT GACTATGATG ATACCATATC 5761 AGTTGAAATG AAGAACGAAG ATTTTGACAT TTATGATGAG GATGAAAATC AGAGCCCCCG 5821 CAGCTTTCAA AAGAAAACAC GACACTATTT TATTGCTGCA GTGGAGAGGC TCTGGGATTA 5881 TGGGATGAGT AGCTCCCCAC ATGTTCTAAG AAACAGGGCT CAGAGTGGCA GTGTCCCTCA 5941 GTTCAAGAAA GTTGTTTTCC AGGAATTTAC TGATCGCTCC TTTACTCAGC CCTTATACCG 6001 TGGAGAACTA AATGAACATT TGGGACTCCT GGGGCCATAT ATAAGAGCAG AAGTTGAAGA 6061 TAATATCATG GTAACTTTCA GAAATCAGGC CTCTCGTCCC TATTCCTTCT ATTCTAGCCT 6121 TATTTCTTAT GAGGAAGATC AGAGGCAAGG AGCAGAACCT AGAAAAAACT TTGTCAAGCC 6181 TAATGAAACC AAAACTTACT TTTGGAAAGT GCAACATCAT ATGGCACCCA CTAAAGATGA 6241 GTTTGACTGC AAAGCCTGGG CTTATTTCTC TGATGTTGAC CTGGAAAAAG ATGTGCACTC 6301 AGGCCTGATT GGACCCCTTC TGGTCTGCCA CACTAACACA CTGAACCCTG CTCATGGGAG 6361 ACAAGTGACA GTACAGGAAT TTGCTCTGTT TTTCACCATC TTTGATGAGA CCAAAAGCTG 6421 GTACTTCACT GAAAATATGG AAAGAAACTG CAGGGCTCCC TGCAATATCC AGATGGAAGA 6481 TCCCACTTTT AAAGAGAATT ATCGCTTCCA TGCAATCAAT GGCTACATAA TGGATACACT 6541 ACCTGGCTTA GTAATGGCTC AGGATCAAAG GATTCGATGG TATCTGCTCA GCATGGGCAG 6601 CAATGAAAAC ATCCATTCTA TTCATTTCAG TGGACATGTG TTCACTGTAC GAAAAAAAGA 6661 GGAGTATAAA ATGGCACTGT ACAATCTCTA TCCAGGTGTT TTTGAGACAG TGGAAATGTT 6721 ACCATCCAAA GCTGGAATTT GGCGGGTGGA ATGCCTTATT GGCGAGCATC TACATSCTSG 6781 GATGAGCACA CTTTTTCTGG TGTACAGCAA TAAGTGTCAG ACTCCCCTGG GAATGGCTTC 6841 TGGACACATT AGAGATTTTC AGATTACAGC TTCAGaACAA TATGGACAGT GGGCCCCAAA 6901 GCTGGCCAGA CTTCATTATT CCGGATCAAT CAATGCCTCC AGCACCAAGG AGCCCTTTTC 6961 TTGGATCAAG GTGGATCTGT TGGCACCAAT GATTATTCAC CGCATCAAGA CCCAGGGTGC 7021 CCGTCAGAAG TTCTCCAGCC TCTACATCTC TCAGTTTATC ATCATGTATA GTCTTGATGG 7081 GAAGAACTGG CAGACTTATC GAGGAAATTC CACTGGAACC TTAATGGTCT TCTTTGGCAA 7141 TGTGGATTCA TCTGCGATAA AACACAATAT TTTTAACCCT CCAATTATTG CTCGATACAT 7201 CCGTTTGCAC CCAACTCATT ATAGCATTCG CAGCACTCTT CGCATGGAGT TGATCCCCTG 7261 TGATTTAAAT AGTTGCAGCA TGCCATTGGG AATGGAGACT AAAGCAATAT CAGATGCACA 7321 GATTACTGCT TCATCCTACT TTACCAATAT GTTTGCCACC TGGTCTCCTT CAAAAGCTCG 7381 ACTTCACCTC CAAGGGAGGA GTAATGCCTG GAGACCTCAG GTGAATAATC CAAAAGAGTG 7441 GCTGCAAGTG GACTTCCAGA AGACAATGAA AGTCACAGGA GTAACTACTC AGGGAGTAAA 7501 ATCTCTGCTT ACCAGCATGT ATGTGAAGGA GTTCCTCATC TCCAGCAGTC AAGATGGCCA 7561 TCAGTGGACT CTCTTTTTTC AGAATGGCAA AGTAAAGGTT TTTCAGGGAA ATCAAGACTC 7621 CTTCACACCT GTGGTGAACT CTCTAGACCC ACCGTTACTG ACTCGCTACC TTCGAATTCA 7681 CCCCCAGAGT TGGGTGCACC AGATTGCCCT GAGGATGGAG GTTCTGGGCT GCGAGGCACA 7741 GGACCTCTAC GACAAAACTC ACACATGCCC ACCGTGCCCA GCTCCAGAAC TCCTGGGCGG 7601 ACCGTCAGTC TTCCTCTTCC CCCCAAAACC CAAGGACACC CTCATGATCT CCCGGACCCC 7861 TGAGGTCACA TGCGTGGTGG TGGACGTGAG CCACGAAGAC CCTGAGGTCA AGTTCAACTG 7921 GTACGTGGAC GGCGTGGAGG TGCATAATGC CAAGACAAAG CCGCGGGAGG AGCAGTACAA 7981 CAGCACGTAC CGTGTGGTCA GCGTCCTCAC CGTCCTGCAC CAGGACTGGC TGAATGGCAA 8041 GGAGTACAAG TGCAAGGTCT CCAACAAAGC CCTCCCAGCC CCCATCGAGA AAACCATCTC 8101 CAAAGCCAAA GGGCAGCCCC GAGAACCACA GGTGTACACC CTGCCCCCAT CCCGGGATGA 8161 GCTGACCAAG AACCAGGTCA GCCTGACCTG CCTGGTCAAA GGCTTCTATC CCAGCGACAT 8221 CGCCGTGGAG TGGGAGAGCA ATGGGCAGCC GGAGAACAAC TACAAGACCA CGCCTCCCGT 8281 GTTGGACTCC GACGGCTCCT TCTTCCTCTA CAGCAAGCTC ACCGTGGACA AGAGCAGGTG 8341 GCAGCAGGGG AACGTCTTCT CATGCTCCGT GATGCATGAG GCTCTGCACA ACCACTACAC 8401 GCAGAAGAGC CTCTCCCTGT CTCCGGGTAA A (ii) Fc (same sequence as A (ii) (SEQ ID NO: 3))] C. B-Domain Deleted FVIII Encoding Nucleotide Sequence (SEQ ID NO: 1) gccaccagaa gatactacct gggtgcagtg gaactgtcat gggactatat gcaaagtgat   60 ctcggtgagc tgcctgtgga cgcaagattt cctcctagag tgccaaaatc ttttccattc  120 aacacctcag tcgtgtacaa aaagactctg tttgtagaat tcacggatca ccttttcaac  180 atcgctaagc caaggccacc ctggatgggt ctgctaggtc ctaccatcca ggctgaggtt  240 tatgatacag tggtcattac acttaagaac atggcttccc atcctgtcag tcttcatgct  300 gttggtgtat cctactggaa agcttctgag ggagctgaat atgatgatca gaccagtcaa  360 agggagaaag aagatgataa agtcttccct ggtggaagcc atacatatgt ctggcaggtc  420 ctgaaagaga atggtccaat ggcctctgac ccactgtgcc ttacctactc atatctttct  480 catgtggacc tggtaaaaga cttgaattca ggcctcattg gagccctact agtatgtaga  540 gaagggagtc tggccaagga aaagacacag accttgcaca aatttatact actttttgct  600 gtatttgatg aagggaaaag ttggcactca gaaacaaaga actccttgat gcaggatagg  660 gatgctgcat ctgctcgggc ctggcctaaa atgcacacag tcaatggtta tgtaaacagg  720 tctctgccag gtctgattgg atgccacagg aaatcagtct attggcatgt gattggaatg  780 ggcaccactc ctgaagtgca ctcaatattc ctcgaaggtc acacatttct tgtgaggaac  840 catcgccagg cgtccttgga aatctcgcca ataactttcc ttactgctca aacactcttg  900 atggaccttg gacagtttct actgttttgt catatctctt cccaccaaca tgatggcatg  960 gaagcttatg tcaaagtaga cagctgtcca gaggaacccc aactacgaat gaaaaataat 1020 gaagaagcgg aagactatga tgatgatctt actgattctg aaatggatgt ggtcaggttt 1080 gatgatgaca actctccttc ctttatccaa attcgctcag ttgccaagaa gcatcctaaa 1140 acttgggtac attacattgc tgctgaagag gaggactggg actatgctcc cttagtcctc 1200 gcccccgatg acagaagtta taaaagtcaa tatttgaaca atggccctca gcggattggt 1260 aggaagtaca aaaaagtccg atttatggca tacacagatg aaacctttaa gactcgtgaa 1320 gctattcagc atgaatcagg aatcttggga cctttacttt atggggaagt tggagacaca 1380 ctgttgatta tatttaagaa tcaagcaagc agaccatata acatctaccc tcacggaatc 1440 actgatgtcc gtcctttgta ttcaaggaga ttaccaaaag gtgtaaaaca tttgaaggat 1500 tttccaattc tgccaggaga aatattcaaa tataaatgga cagtgactgt agaagatggg 1560 ccaactaaat cagatcctcg gtgcctgacc cgctattact ctagtttcgt taatatggag 1620 agagatctag cttcaggact cattggccct ctcctcatct gctacaaaga atctgtagat 1680 caaagaggaa accagataat gtcagacaag aggaatgtca tcctgttttc tgtatttgat 1740 gagaaccgaa gctggtacct cacagagaat atacaacgct ttctccccaa tccagctgga 1800 gtgcagcttg aggatccaga gttccaagcc tccaacatca tgcacagcat caatggctat 1860 gtttttgata gtttgcagtt gtcagtttgt ttgcatgagg tggcatactg gtacattcta 1920 agcattggag cacagactga cttcctttct gtcttcttct ctggatatac cttcaaacac 1980 aaaatggtct atgaagacac actcacccta ttcccattct caggagaaac tgtcttcatg 2040 tcgatggaaa acccaggtct atggattctg gggtgccaca actcagactt tcggaacaga 2100 ggcatgaccg ccttactgaa ggtttctagt tgtgacaaga acactggtga ttattacgag 2160 gacagttatg aagatatttc agcatacttg ctgagtaaaa acaatgccat tgaaccaaga 2220 agcttctctc aaaacccacc agtcttgaaa cgccatcaac gggaaataac tcgtactact 2280 cttcagtcag atcaagagga aattgactat gatgatacca tatcagttga aatgaagaag 2340 gaagattttg acatttatga tgaggatgaa aatcagagcc cccgcagctt tcaaaagaaa 2400 acacgacact attttattgc tgcagtggag aggctctggg attatgggat gagtagctcc 2460 ccacatgttc taagaaacag ggctcagagt ggcagtgtcc ctcagttcaa gaaagttgtt 2520 ttccaggaat ttactgatgg ctcctttact cagcccttat accgtggaga actaaatgaa 2380 catttgggac tcctggggcc atatataaga gcagaagttg aagataatat catggtaact 2640 ttcagaaatc aggcctctcg tccctattcc ttctattcta gccttatttc ttatgaggaa 2700 gatcagaggc aaggagcaga acctagaaaa aactttgtca agcctaatga aaccaaaact 2760 tacttttgga aagtgcaaca tcatatggca cccactaaag atgagtttga ctgcaaagcc 2820 tgggcttatt tctctgatgt tgacctggaa aaagatgtgc actcaggcct gattggaccc 2880 cttctggtct gccacactaa cacactgaac cctgctcatg ggagacaagt gacagtacag 2940 gaatttgctc tgtttttcac catctttgat gagaccaaaa gctggtactt cactgaaaat 3000 atggaaagaa actgcagggc tccctgcaat atccagatgg aagatcccac ttttaaagag 3060 aattatcgct tccatgcaat caatggctac ataatggata cactacctgg cttagtaatg 3120 gctcaggatc aaaggattcg atggtatctg ctcagcatgg gcagcaatga aaacatccat 3180 tctattcatt tcagtggaca tgtgttcact gtacgaaaaa aagaggagta taaaatggca 3240 ctgtacaatc tctatccagg tgtttttgag acagtggaaa tgttaccatc caaagctgga 3300 atttggcggg tggaatgcct tattggcgag catctacatg ctgggatgag cacacttttt 3360 ctggtgtaca gcaataagtg tcagactccc ctgggaatgg cttctggaca cattagagat 3420 tttcagatta cagcttcagg acaatatgga cagtgggccc caaagctggc cagacttcat 3480 tattccggat caatcaatgc ctggagcacc aaggagccct tttcttggat caaggtggat 3540 ctgttggcac caatgattat tcacggcatc aagacccagg gtgcccgtca gaagttctcc 3600 agcctctaca tctctcagtt tatcatcatg tatagtcttg atgggaagaa gtggcagact 3660 tatcgaggaa attccactgg aaccttaatg gtcttctttg gcaatgtgga ttcatctggg 3720 ataaaacaca atatttttaa ccctccaatt attgctcgat acatccgttt gcacccaact 3780 cattatagca ttcgcagcac tcttcgcatg gagttgatgg gctgtgattt aaatagttgc 3840 agcatgccat tgggaatgga gagtaaagca atatcagatg cacagattac tgcttcatcc 3900 tactttacca atatgtttgc cacctggtct ccttcaaaag ctcgacttca cctccaaggg 3960 aggagtaatg cctggagacc tcaggtgaat aatccaaaag agtggctgca agtggacttc 4020 cagaagacaa tgaaagtcac aggagtaact actcagggag taaaatctct gcttaccagc 4080 atgtatgtga aggagttcct catctccagc agtcaagatg gccatcagtg gactctcttt 4140 tttcagaatg gcaaagtaaa ggtttttcag ggaaatcaag actccttcac acctgtggtg 4200 aactctctag acccaccgtt actgactcgc taccttcgaa ttcaccccca gagttgggtg 4260 caccagattg ccctgaggat ggaggttctg ggctgcgagg cacaggacct ctac 4314 D. Full-length FVIII Encoding Nucleotide Sequence (SEQ ID NO: 5) gccaccagaa gatactacct gggtgcagtg gaactgtcat gggactatat gcaaagtgat   60 ctcggtgagc tgcctgtgga cgcaagattt cctcctagag tgccaaaatc ttttccattc  120 aacacctcag tcgtgtacaa aaagactctg tttgtagaat tcacggatca ccttttcaac  180 atcgctaagc caaggccacc ctggatgggt ctgctaggtc ctaccatcca ggctgaggtt  240 tatgatacag tggtcattac acttaagaac atggcttccc atcctgtcag tcttcatgct  300 gttggtgtat cctactggaa agcttctgag ggagctgaat atgatgatca gaccagtcaa  360 agggagaaag aagatgataa agtcttccct ggtggaagcc atacatatgt ctggcaggtc  420 ctgaaagaga atggtccaat ggcctctgac ccactgtgcc ttacctactc atatctttct  480 catgtggacc tggtaaaaga cttgaattca ggcctcattg gagccctact agtatgtaga  540 gaagggagtc tggccaagga aaagacacag accttgcaca aatttatact actttttgct  600 gtatttgatg aagggaaaag ttggcactca gaaacaaaga actccttgat gcaggatagg  660 gatgctgcat ctgctcgggc ctggcctaaa atgcacacag tcaatggtta tgtaaacagg  720 tctctgccag gtctgattgg atgccacagg aaatcagtct attggcatgt gattggaatg  780 ggcaccactc ctgaagtgca ctcaatattc ctcgaaggtc acacatttct tgtgaggaac  840 catcgccagg cgtccttgga aatctcgcca ataactttcc ttactgctca aacactcttg  900 atggaccttg gacagtttct actgttttgt catatctctt cccaccaaca tgatggcatg  960 gaagcttatg tcaaagtaga cagctgtcca gaggaacccc aactacgaat gaaaaataat 1020 gaagaagcgg aagactatga tgatgatctt actgattctg aaatggatgt ggtcaggttt 1080 gatgatgaca actctccttc ctttatccaa attcgctcag ttgccaagaa gcatcctaaa 1140 acttgggtac attacattgc tgctgaagag gaggactggg actatgctcc cttagtcctc 1200 gcccccgatg acagaagtta taaaagtcaa tatttgaaca atggccctca gcggattggt 1260 aggaagtaca aaaaagtccg atttatggca tacacagatg aaacctttaa gactcgtgaa 1320 gctattcagc atgaatcagg aatcttggga cctttacttt atggggaagt tggagacaca 1380 ctgttgatta tatttaagaa tcaagcaagc agaccatata acatctaccc tcacggaatc 1440 actgatgtcc gtcctttgta ttcaaggaga ttaccaaaag gtgtaaaaca tttgaaggat 1500 tttccaattc tgccaggaga aatattcaaa tataaatgga cagtgactgt agaagatggg 1560 ccaactaaat cagatcctcg gtgcctgacc cgctattact ctagtttcgt taatatggag 1620 agagatctag cttcaggact cattggccct ctcctcatct gctacaaaga atctgtagat 1680 caaagaggaa accagataat gtcagacaag aggaatgtca tcctgttttc tgtatttgat 1740 gagaaccgaa gctggtacct cacagagaat atacaacgct ttctccccaa tccagctgga 1800 gtgcagcttg aggatccaga gttccaagcc tccaacatca tgcacagcat caatggctat 1860 gtttttgata gtttgcagtt gtcagtttgt ttgcatgagg tggcatactg gtacattcta 1920 agcattggag cacagactga cttcctttct gtcttcttct ctggatatac cttcaaacac 1980 aaaatggtct atgaagacac actcacccta ttcccattct caggagaaac tgtcttcatg 2040 tcgatggaaa acccaggtct atggattctg gggtgccaca actcagactt tcggaacaga 2100 ggcatgaccg ccttactgaa ggtttctagt tgtgacaaga acactggtga ttattacgag 2160 gacagttatg aagatatttc agcatacttg ctgagtaaaa acaatgccat tgaaccaaga 2220 agcttctccc agaattcaag acaccctagc actaggcaaa agcaatttaa tgccaccaca 2280 attccagaaa atgacataga gaagactgac ccttggtttg cacacagaac acctatgcct 2340 aaaatacaaa atgtctcctc tagtgatttg ttgatgctct tgcgacagag tcctactcca 2400 catgggctat ccttatctga tctccaagaa gccaaatatg agactttttc tgatgatcca 2460 tcacctggag caatagacag taataacagc ctgtctgaaa tgacacactt caggccacag 2520 ctccatcaca gtggggacat ggtatttacc cctgagtcag gcctccaatt aagattaaat 2580 gagaaactgg ggacaactgc agcaacagag ttgaagaaac ttgatttcaa agtttctagt 2640 acatcaaata atctgatttc aacaattcca tcagacaatt tggcagcagg tactgataat 2700 acaagttcct taggaccccc aagtatgcca gttcattatg atagtcaatt agataccact 2760 ctatttggca aaaagtcatc tccccttact gagtctggtg gacctctgag cttgagtgaa 2820 gaaaataatg attcaaagtt gttagaatca ggtttaatga atagccaaga aagttcatgg 2880 ggaaaaaatg tatcgtcaac agagagtggt aggttattta aagggaaaag agctcatgga 2940 cctgctttgt tgactaaaga taatgcctta ttcaaagtta gcatctcttt gttaaagaca 3000 aacaaaactt ccaataattc agcaactaat agaaagactc acattgatgg cccatcatta 3060 ttaattgaga atagtccatc agtctggcaa aatatattag aaagtgacac tgagtttaaa 3120 aaagtgacac ctttgattca tgacagaatg cttatggaca aaaatgctac agctttgagg 3180 ctaaatcata tgtcaaataa aactacttca tcaaaaaaca tggaaatggt ccaacagaaa 3240 aaagagggcc ccattccacc agatgcacaa aatccagata tgtcgttctt taagatgcta 3300 ttcttgccag aatcagcaag gtggatacaa aggactcatg gaaagaactc tctgaactct 3360 gggcaaggcc ccagtccaaa gcaattagta tccttaggac cagaaaaatc tgtggaaggt 3420 cagaatttct tgtctgagaa aaacaaagtg gtagtaggaa agggtgaatt tacaaaggac 3480 gtaggactca aagagatggt ttttccaagc agcagaaacc tatttcttac taacttggat 3540 aatttacatg aaaataatac acacaatcaa gaaaaaaaaa ttcaggaaga aatagaaaag 3600 aaggaaacat taatccaaga gaatgtagtt ttgcctcaga tacatacagt gactggcact 3660 aagaatttca tgaagaacct tttcttactg agcactaggc aaaatgtaga aggttcatat 3720 gacggggcat atgctccagt acttcaagat tttaggtcat taaatgattc aacaaataga 3780 acaaagaaac acacagctca tttctcaaaa aaaggggagg aagaaaactt ggaaggcttg 3840 ggaaatcaaa ccaagcaaat tgtagagaaa tatgcatgca ccacaaggat atctcctaat 3900 acaagccagc agaattttgt cacgcaacgt agtaagagag ctttgaaaca attcagactc 3960 ccactagaag aaacagaact tgaaaaaagg ataattgtgg atgacacctc aacccagtgg 4020 tccaaaaaca tgaaacattt gaccccgagc accctcacac agatagacta caatgagaag 4080 gagaaagggg ccattactca gtctccctta tcagattgcc ttacgaggag tcatagcatc 4140 cctcaagcaa atagatctcc attacccatt gcaaaggtat catcatttcc atctattaga 4200 cctatatatc tgaccagggt cctattccaa gacaactctt ctcatcttcc agcagcatct 4260 tatagaaaga aagattctgg ggtccaagaa agcagtcatt tcttacaagg agccaaaaaa 4320 aataaccttt ctttagccat tctaaccttg gagatgactg gtgatcaaag agaggttggc 4380 tccctgggga caagtgccac aaattcagtc acatacaaga aagttgagaa cactgttctc 4440 ccgaaaccag acttgcccaa aacatctggc aaagttgaat tgcttccaaa agttcacatt 4500 tatcagaagg acctattccc tacggaaact agcaatgggt ctcctggcca tctggatctc 4560 gtggaaggga gccttcttca gggaacagag ggagcgatta agtggaatga agcaaacaga 4620 cctggaaaag ttccctttct gagagtagca acagaaagct ctgcaaagac tccctccaag 4680 ctattggatc ctcttgcttg ggataaccac tatggtactc agataccaaa agaagagtgg 4740 aaatcccaag agaagtcacc agaaaaaaca gcttttaaga aaaaggatac cattttgtcc 4800 ctgaacgctt gtgaaagcaa tcatgcaata gcagcaataa atgagggaca aaataagccc 4860 gaaatagaag tcacctgggc aaagcaaggt aggactgaaa ggctgtgctc tcaaaaccca 4920 ccagtcttga aacgccatca acgggaaata actcgtacta ctcttcagtc agatcaagag 4980 gaaattgact atgatgatac catatcagtt gaaatgaaga aggaagattt tgacatttat 5040 gatgaggatg aaaatcagag cccccgcagc tttcaaaaga aaacacgaca ctattttatt 5100 gctgcagtgg agaggctctg ggattatggg atgagtagct ccccacatgt tctaagaaac 5160 agggctcaga gtggcagtgt ccctcagttc aagaaagttg ttttccagga atttactgat 5220 ggctccttta ctcagccctt ataccgtgga gaactaaatg aacatttggg actcctgggg 5280 ccatatataa gagcagaagt tgaagataat atcatggtaa ctttcagaaa tcaggcctct 5340 cgtccctatt ccttctattc tagccttatt tcttatgagg aagatcagag gcaaggagca 5400 gaacctagaa aaaactttgt caagcctaat gaaaccaaaa cttacttttg gaaagtgcaa 5460 catcatatgg cacccactaa agatgagttt gactgcaaag cctgggctta tttctctgat 5520 gttgacctgg aaaaagatgt gcactcaggc ctgattggac cccttctggt ctgccacact 5580 aacacactga accctgctca tgggagacaa gtgacagtac aggaatttgc tctgtttttc 5640 accatctttg atgagaccaa aagctggtac ttcactgaaa atatggaaag aaactgcagg 5700 gctccctgca atatccagat ggaagatccc acttttaaag agaattatcg cttccatgca 5760 atcaatggct acataatgga tacactacct ggcttagtaa tggctcagga tcaaaggatt 5820 cgatggtatc tgctcagcat gggcagcaat gaaaacatcc attctattca tttcagtgga 5880 catgtgttca ctgtacgaaa aaaagaggag tataaaatgg cactgtacaa tctctatcca 5940 ggtgtttttg agacagtgga aatgttacca tccaaagctg gaatttggcg ggtggaatgc 6000 cttattggcg agcatctaca tgctgggatg agcacacttt ttctggtgta cagcaataag 6060 tgtcagactc ccctgggaat ggcttctgga cacattagag attttcagat tacagcttca 6120 ggacaatatg gacagtgggc cccaaagctg gccagacttc attattccgg atcaatcaat 6160 gcctggagca ccaaggagcc cttttcttgg atcaaggtgg atctgttggc accaatgatt 6240 attcacggca tcaagaccca gggtgcccgt cagaagttct ccagcctcta catctctcag 6300 tttatcatca tgtatagtct tgatgggaag aagtggcaga cttatcgagg aaattccact 6360 ggaaccttaa tggtcttctt tggcaatgtg gattcatctg ggataaaaca caatattttt 6420 aaccctccaa ttattgctcg atacatccgt ttgcacccaa ctcattatag cattcgcagc 6480 actcttcgca tggagttgat gggctgtgat ttaaatagtt gcagcatgcc attgggaatg 6540 gagagtaaag caatatcaga tgcacagatt actgattcat cctactttac caatatgttt 6600 gccacctggt ctccttcaaa agctcgactt cacctccaag ggaggagtaa tgcctggaga 6660 cctcaggtga ataatccaaa agagtggctg caagtggact tccagaagac aatgaaagtc 6720 acaggagtaa ctactcaggg agtaaaatct ctgcttacca gcatgtatgt gaaggagttc 6780 ctcatctcca gcagtcaaga tggccatcag tggactctct tttttcagaa tggcaaagta 6840 aaggtttttc agggaaatca agactccttc acacctgtgg tgaactctct agacccaccg 6900 ttactgactc gctaccttcg aattcacccc cagagttggg tgcaccagat tgccctgagg 6960 atggaggttc tgggctgcga ggcacaggac ctctac 6996

TABLE 2 Polypeptide Sequences A. B-Domain Deleted FVIII-Fc Monomer Hybrid: created by coexpressing BDD FVIIIFc and Fc chains. Construct = HC-LC-Fc fusion. An Fc expression cassette is cotransfected with BDDFVIII-Fc to generate the BDD FVIIIFc monomer-. For the BDD FVIIIFc chain, the Fc sequence is shown in bold; HC sequence is shown in double underline; remaining B domain sequence is shown in italics. Signal peptides are underlined. i) B domain deleted FVIII-Fc chain (19 amino acid signal sequence underlined) (SEQ ID NO: 60) MQIELSTCFFLCLLRFCFS ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPR PPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFP GGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKF ILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGM GTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVK VDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEE EDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREATQHESGILGPLLYG EVGDTLLIIFKNQASRPYNIYPHCITDVRPLYSRRLPKGVKHLKETPILPGEIFKYKWTVTVEDG PTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSW YLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYTLSIGAQTDFLS VFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNT GDYYEDSYEDISAYLLSKNNATEPR SFSQAMPVLERHQREITRTTLQSDQEEIDYDDTISVEMKK EDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFT DGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRK NFVKPNETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGR QVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGINM AQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVE CLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWST KEPFSWIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGN VDSSGIKHNIFNETITARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLCMESKAISDAQTTASS YFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKE FLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVL GCEAQDLYDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCAVSNKALPAPIEKTISKAKGQ PREPQVYTLPPSRDELTKNQVSLTCLVIMFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLY SKLTVDICSRWQQGNVFSCSVMHEALHNHYTQWLSLSPGK ii) Fc chain (20 amino acid heterologous signal peptide from mouse Igκ chain underlined) (SEQ ID NO: 4) METDTLLLWVLLLWVPGSTG DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALRAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK ii) B-Domain Deleted FVIII Without Signal Peptide (SEQ ID NO: 2) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPR PPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTSQREKEDDKVFP GGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKF ILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWHVIGM GTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVK VDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEE EDWDYAPLVLAPEQRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLZYG EVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIFKYKWTVTVEDG PTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSW YLTENIQRFLPNPAGVQLED2EFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLS VFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKVSSCDKNT GDYYEDSYEDISAYLLSKNNAIEPRSFSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKK EDFDIYDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFT DGSFTQPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRK NFVKPNETKTYFWKVQEHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGR QVTVQEFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVM AQDQRIRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVE CLIGEHLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSQSINAWST KEPFSWIKVDLLAPMITHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGN VDSSGIKHNIFNPPIIARYIRLHPTEYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASS YFTNMFATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKE FLISSSQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVL GCEAQDLY B. Full-length FVIIIFc monomer hybrid: created by coexpressing FVIIIFc and Fc chains. Construct = HC-B-LC-Fc fusion. An Fc expression cassette is cotransfected with full-length FVIII-Fc to generate the full-length FVIIIFc monomer. For the FVIIIFc chain, the Fc sequence is shown in bold; HC sequence is shown in double underline; B domain sequence is shown in italics. Signal peptides are underlined. i) Full-length FVIIIFc chain (FVIII signal peptide underlined (SEQ ID NO: 62) MQIELSTCFFLCLLRFCFS ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEFTDHLFNIAKPR PPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQISQREKEDDKVFP GGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSLAKEKTQTLHKF ILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCHRKSVYWAVIGM GTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISSHQHDGMEAYVK VDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHPKTWVHYIAAEE EDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQHESGILGPLLYG EVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFFILEGEIFKYKWTVTVEDG PTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVILFSVFDENRSW YLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYILSIGAQTDFLS VFFSGYTFKHKMVYEDTLTLFETSGETVFMSMENPGLWILGCHNSDFRNRGMTALLKySSQDKNT GDYYEDSYEDISAYLLSKNNAIEPR SFSQNSRHPSTRQKQFNATTIPENDIEKTDPWFAHRTPMP KIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEMTHFRPQLHHSG DMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTDNTSSLGPPSMP VHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNVSSTESGRLFKG KRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVWQNILESDTEFK KVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDMSFFKMLFLPES ARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTKDVGLKEMVFPS SRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFMKNLFLLSTRQN VEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVEKYACTTRISPN TSQQNFVTQRSKRALKQFRLFLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQIDYNEKEKGAI TQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAASYRKKDSGVQE SSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPDLPKTSGKVELL PKVHIYQKDLFPTETSNGSPGHLDLVEGSLLQGTEGAIKWNEANRPGKVPFLRVATESSAKTPSK LLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAINEGQNKPEIEVT WAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDIYDEDENQSPRS FQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFTQPLYRGELNEH LGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKPNETKTYFWKVQ HHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQEFALFFTIFDE TKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQRIRWYLLSMGSN ENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGEHLHAGMSTLFL VYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFSWIKVDLLAPMI IHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSGIKHNIFNPPII ARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNMFATWSPSKARL HLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISSSQDGHQWTLFF QNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQDLYDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVENAKTKPRE EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDEL TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVF SCSVMHEALHNHYTQKSLSLSPGK ii) Fc chain (20 amino acid heterologous signal peptide from mouse Igκ chain underlined) (SEQ ID NO: 4) METDTLLLWVLLLWVPGSTG DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK iii) Full-leugth FVIII Without Signal Peptide (SEQ ID NO: 6) ATRRYYLGAVELSWDYMQSDLGELPVDARFPPRVPKSFPFNTSVVYKKTLFVEF TDHLFNIAKPREPWMGLLGPTIQAEVYDTVVITLKNMASHPVSLHAVGVSYWKASEGAEYDDQTS QREKEDDKVFPGGSHTYVWQVLKENGPMASDPLCLTYSYLSHVDLVKDLNSGLIGALLVCREGSL AKEKTQTLHKFILLFAVFDEGKSWHSETKNSLMQDRDAASARAWPKMHTVNGYVNRSLPGLIGCH RKSVYWHVIGMGTTPEVHSIFLEGHTFLVRNHRQASLEISPITFLTAQTLLMDLGQFLLFCHISS HQHDGMEAYVKVDSCPEEPQLRMKNNEEAEDYDDDLTDSEMDVVRFDDDNSPSFIQIRSVAKKHP KTWVHYIAAEEEDWDYAPLVLAPDDRSYKSQYLNNGPQRIGRKYKKVRFMAYTDETFKTREAIQH ESGILGPLLYGEVGDTLLIIFKNQASRPYNIYPHGITDVRPLYSRRLPKGVKHLKDFPILPGEIF KYKWTVTVEDGPTKSDPRCLTRYYSSFVNMERDLASGLIGPLLICYKESVDQRGNQIMSDKRNVI LFSVFDENRSWYLTENIQRFLPNPAGVQLEDPEFQASNIMHSINGYVFDSLQLSVCLHEVAYWYI LSIGAQTDFLSVFFSGYTFKHKMVYEDTLTLFPFSGETVFMSMENPGLWILGCHNSDFRNRGMTA LLKVSSCDKNTGDYYEDSYEDISAYLLSKNNAIEPRSFSQNSRHPSTRQKQFNATTIPENDIEKT DPWFAHRTPMPKIQNVSSSDLLMLLRQSPTPHGLSLSDLQEAKYETFSDDPSPGAIDSNNSLSEM THFRPQLHHSGDMVFTPESGLQLRLNEKLGTTAATELKKLDFKVSSTSNNLISTIPSDNLAAGTD NTSSLGPPSMPVHYDSQLDTTLFGKKSSPLTESGGPLSLSEENNDSKLLESGLMNSQESSWGKNV SSTESGRLFKGKRAHGPALLTKDNALFKVSISLLKTNKTSNNSATNRKTHIDGPSLLIENSPSVW QNILESDTEFKKVTPLIHDRMLMDKNATALRLNHMSNKTTSSKNMEMVQQKKEGPIPPDAQNPDM SFFKMLFLPESARWIQRTHGKNSLNSGQGPSPKQLVSLGPEKSVEGQNFLSEKNKVVVGKGEFTK DVGLKEMVFPSSRNLFLTNLDNLHENNTHNQEKKIQEEIEKKETLIQENVVLPQIHTVTGTKNFM KNLFLLSTRQNVEGSYDGAYAPVLQDFRSLNDSTNRTKKHTAHFSKKGEEENLEGLGNQTKQIVE KYACTTRISPNTSQQNFVTQRSKRALKQFRLPLEETELEKRIIVDDTSTQWSKNMKHLTPSTLTQ IDYNEKEKGAITQSPLSDCLTRSHSIPQANRSPLPIAKVSSFPSIRPIYLTRVLFQDNSSHLPAA SYRKKDSGVQESSHFLQGAKKNNLSLAILTLEMTGDQREVGSLGTSATNSVTYKKVENTVLPKPD LPKTSGKVELLPKVHIYQKDLFPTETSNGSPGELDLVEGSLLQGTEGAIKWNEANRPGKVPFLRV ATESSAKTPSKLLDPLAWDNHYGTQIPKEEWKSQEKSPEKTAFKKKDTILSLNACESNHAIAAIN EGQNKPEIEVTWAKQGRTERLCSQNPPVLKRHQREITRTTLQSDQEEIDYDDTISVEMKKEDFDI YDEDENQSPRSFQKKTRHYFIAAVERLWDYGMSSSPHVLRNRAQSGSVPQFKKVVFQEFTDGSFT QPLYRGELNEHLGLLGPYIRAEVEDNIMVTFRNQASRPYSFYSSLISYEEDQRQGAEPRKNFVKP NETKTYFWKVQHHMAPTKDEFDCKAWAYFSDVDLEKDVHSGLIGPLLVCHTNTLNPAHGRQVTVQ EFALFFTIFDETKSWYFTENMERNCRAPCNIQMEDPTFKENYRFHAINGYIMDTLPGLVMAQDQR IRWYLLSMGSNENIHSIHFSGHVFTVRKKEEYKMALYNLYPGVFETVEMLPSKAGIWRVECLIGE HLHAGMSTLFLVYSNKCQTPLGMASGHIRDFQITASGQYGQWAPKLARLHYSGSINAWSTKEPFS WIKVDLLAPMIIHGIKTQGARQKFSSLYISQFIIMYSLDGKKWQTYRGNSTGTLMVFFGNVDSSG IKHNIFNPPIIARYIRLHPTHYSIRSTLRMELMGCDLNSCSMPLGMESKAISDAQITASSYFTNM FATWSPSKARLHLQGRSNAWRPQVNNPKEWLQVDFQKTMKVTGVTTQGVKSLLTSMYVKEFLISS SQDGHQWTLFFQNGKVKVFQGNQDSFTPVVNSLDPPLLTRYLRIHPQSWVHQIALRMEVLGCEAQ DLY 

1. A method for decreasing nonprocessed Factor VIII in a culturing medium, comprising, a) expressing Factor VIII from a first polynucleotide in a host cell, where the endogenous processing enzymes of the host cell are insufficient to convert all of the Factor VIII to its processed isoform; b) contacting said Factor VIII with an exogenous proprotein convertase; and c) decreasing the nonprocessed Factor VIII by processing with said proprotein convertase.
 2. The method of claim 1, wherein said exogenous proprotein convertase is expressed from a second polypeptide in the host cell expressing said Factor VIII or is added to said culture medium. 3-4. (canceled)
 5. The method of claim 1, wherein said Factor VIII is processed by cleaving Arginine corresponding to amino acid residue 1648 in SEQ ID NO: 6 (full-length Factor VIII) or amino acid residue 754 in SEQ ID NO: 2 (B domain-deleted Factor VIII).
 6. The method of claim 5, wherein said processed Factor VIII comprises a Factor VIII heavy chain and a Factor VIII light chain, wherein said heavy chain and light chain are associated by a non-covalent bond. 7-8. (canceled)
 9. The method of claim 1, wherein said proprotein convertase is selected from the group consisting of proprotein convertase subtilisin/kexin type 3, proprotein convertase subtilisin/kexin type 5, proprotein convertase subtilisin/kexin type 7, a yeast Kex 2 and a combination thereof. 10-19. (canceled)
 20. The method of claim 1, wherein said Factor VIII is expressed as full-length Factor VIII or partial or full B-domain deleted Factor VIII. 21-28. (canceled)
 29. The method of claim 1, wherein said Factor VIII is fused to an immunoglobulin constant region or a portion thereof. 30-34. (canceled)
 35. The method of claim 1, wherein said first polynucleotide and said second polynucleotide are located on the same vector or on two different vectors. 36-39. (canceled)
 40. The method of claim 1, wherein said host cell is a CHO cell, a HEK293 cell, a HKB11 cell, or a BHK cell.
 41. A host cell comprising an expression vector comprising a first polynucleotide sequence encoding Factor VIII and a second polynucleotide sequence encoding a proprotein convertase, wherein said Factor VIII is processed by said proprotein convertase.
 42. The host cell of claim 41, wherein said first and second polynucleotides are contained in a first and second expression vectors, respectively.
 43. The host cell of claim 41, wherein said proprotein convertase cleaves Factor VIII at Arginine located at corresponding to amino acid residue 1648 in SEQ ID NO: 6 (full-length Factor VIII) or amino acid residue 754 in SEQ ID NO: 2 (BDD Factor VIII).
 44. The host cell of claim 41, wherein said Factor VIII contains more than 75%, 80%, 85%, 90%, 95%, and 100% of the processed Factor VIII after processing by said proprotein convertase. 45-47. (canceled)
 48. The host cell of claim 41, wherein said proprotein convertase is selected from the group consisting of proprotein convertase subtilisin/kexin type 3, proprotein convertase subtilisin/kexin type 5, proprotein convertase subtilisin/kexin type 7, a yeast Kex 2 and a combination thereof. 49-67. (canceled)
 68. The host cell of claim 41, wherein said host cell is a CHO cell, a HEK293 cell, a HKB11 cell, or a BHK cell.
 69. A method of producing Factor VIII, comprising a) culturing the host cell of claim 41 in a culture medium that expresses said Factor VIII and said proprotein convertase, and b) processing said Factor VIII by said protein convertase.
 70. (canceled)
 71. An expression vector comprising a first polynucleotide encoding Factor VIII and a second polynucleotide encoding a proprotein convertase, wherein said Factor VIII is processed by the proprotein convertase when said Factor VIII and said proprotein convertase are expressed in a host cell comprising said vector.
 72. The vector of claim 71, wherein said proprotein convertase cleaves Factor VIII at Arginine corresponding to amino acid residue 1648 in SEQ ID NO: 6 (full-length Factor VIII) or amino acid residue 754 in SEQ ID NO: 2 (B-domain deleted Factor VIII) when said proprotein convertase is expressed in a host cell comprising said vector. 73-86. (canceled)
 87. A method of producing Factor VIII, comprising a) transfecting the expression vector of claim 71 in a host cell, b) culturing said host cell in a culture medium to express said Factor VIII and said proprotein convertase, wherein said proprotein convertase processes said Factor VIII.
 88. (canceled)
 89. An isolated polypeptide comprising Factor VIII produced by the method of claim
 1. 